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The majority of inherited retinal degenerations converge on the phenotype of photoreceptor cell death. Second- and third-order neurons are spared in these diseases, making it possible to restore retinal light responses using optogenetics. Viral expression of channelrhodopsin in the third-order neurons under ubiquitous promoters was previously shown to restore visual function, albeit at light intensities above illumination safety thresholds. Here, we report (to our knowledge, for the first time) activation of macaque retinas, up to 6 months post-injection, using channelrhodopsin-Ca2+-permeable channelrhodopsin (CatCh) at safe light intensities. High-level CatCh expression was achieved due to a new promoter based on the regulatory region of the gamma-synuclein gene (SNCG) allowing strong expression in ganglion cells across species. Our promoter, in combination with clinically proven adeno-associated virus 2 (AAV2), provides CatCh expression in peri-foveolar ganglion cells responding robustly to light under the illumination safety thresholds for the human eye. On the contrary, the threshold of activation and the proportion of unresponsive cells were much higher when a ubiquitous promoter (cytomegalovirus [CMV]) was used to express CatCh. The results of our study suggest that the inclusion of optimized promoters is key in the path to clinical translation of optogenetics.

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Neural transplantation is a promising therapeutic approach for neurodegenerative diseases; however, many patients receiving intracerebral fetal allografts exhibit signs of immunization to donor antigens that could compromise the graft. In this context, we intracerebrally transplanted mesencephalic pig xenografts into primates to identify a suitable strategy to enable long‐term cell survival, maturation, and differentiation. Parkinsonian primates received WT or CTLA4‐Ig transgenic porcine xenografts and different durations of peripheral immunosuppression to test whether systemic plus graft‐mediated local immunosuppression might avoid rejection. A striking recovery of spontaneous locomotion was observed in primates receiving systemic plus local immunosuppression for 6 mo. Recovery was associated with restoration of dopaminergic activity detected both by positron emission tomography imaging and histological examination. Local infiltration by T cells and CD80/86+ microglial cells expressing indoleamine 2,3‐dioxigenase were observed only in CTLA4‐Ig recipients. Results suggest that in this primate neurotransplantation model, peripheral immunosuppression is indispensable to achieve the long‐term survival of porcine neuronal xenografts that is required to study the beneficial immunomodulatory effect of local blockade of T cell costimulation. The intragraft expression of CTLA4‐Ig, a costimulation blocker, and the duration of the peripheral immunosuppressive treatment can improve cell survival and delay local xenogeneic T cell activation following intracerebral transplantation of transgenic CTLA4‐Ig+/+ pig xenografts in parkinsonian nonhuman primates.

Spliceosome‐mediated RNAtrans‐splicing, or SMaRT, is a promising strategy to design innovative gene therapy solutions for currently intractable genetic diseases. SMaRTrelies on the correction of mutations at the post‐transcriptional level by modifying the mRNAsequence. To achieve this, an exogenous RNAis introduced into the target cell, usually by means of gene transfer, to induce a splice event in transbetween the exogenous RNAand the target endogenous pre‐mRNA. This produces a chimeric mRNAcomposed partly of exons of the latter, and partly of exons of the former, encoding a sequence free of mutations. The principal challenge of SMaRTtechnology is to achieve a reaction as complete as possible, i.e., resulting in 100% repairing of the endogenous mRNAtarget. The proof of concept of SMaRTfeasibility has already been established in several models of genetic diseases caused by recessive mutations. In such cases, in fact, the repair of only a portion of the mutant mRNApool may be sufficient to obtain a significant therapeutic effect. However in the case of dominant mutations, the target cell must be freed from the majority of mutant mRNAcopies, requiring a highly efficient trans‐splicing reaction. This likely explains why only a few examples of SMaRTapproaches targeting dominant mutations are reported in the literature. In this review, we explain in details the mechanism of trans‐splicing, review the different strategies that are under evaluation to lead to efficient trans‐splicing, and discuss the advantages and limitations of SMaRT. WIREs RNA2016, 7:487–498. doi: 10.1002/wrna.1347 For further resources related to this article, please visit the WIREs website.

Neural transplantation is a promising therapeutic approach for neurodegenerative diseases; however, many patients receiving intracerebral fetal allografts exhibit signs of immunization to donor antigens that could compromise the graft. In this context, we intracerebrally transplanted mesencephalic pig xenografts into primates to identify a suitable strategy to enable long‐term cell survival, maturation, and differentiation. Parkinsonian primates received WT or CTLA4‐Ig transgenic porcine xenografts and different durations of peripheral immunosuppression to test whether systemic plus graft‐mediated local immunosuppression might avoid rejection. A striking recovery of spontaneous locomotion was observed in primates receiving systemic plus local immunosuppression for 6 mo. Recovery was associated with restoration of dopaminergic activity detected both by positron emission tomography imaging and histological examination. Local infiltration by T cells and CD80/86+ microglial cells expressing indoleamine 2,3‐dioxigenase were observed only in CTLA4‐Ig recipients. Results suggest that in this primate neurotransplantation model, peripheral immunosuppression is indispensable to achieve the long‐term survival of porcine neuronal xenografts that is required to study the beneficial immunomodulatory effect of local blockade of T cell costimulation.

The discovery of the important role of cerebrospinal fluid (CSF) drainage of cerebral metabolite waste, known as the glymphatic system, has changed our view of brain waste clearance. We recently performed experiments to evaluate the glymphatic system in non-human primates (NHP). Here, we report the case of an NHP with iatrogenic CSF leakage. In this animal, solute transport through the brain, assessed by gadolinium injection in the CSF, was severely impaired by iatrogenic pseudomeningocele. This observation raises an important question: does brain surgery, and particularly posterior fossa surgery, lead to chronic impairment of parenchymal CSF circulation and solute transport?

Considerable progress has been made in generating fully functional and transplantable dopamine neurons from human embryonic stem cells (hESCs). Before these cells can be used for cell replacement therapy in Parkinson’s disease (PD), it is important to verify their functional properties and efficacy in animal models. Here we provide a comprehensive preclinical assessment of hESC-derived midbrain dopamine neurons in a rat model of PD. We show long-term survival and functionality using clinically relevant MRI and PET imaging techniques and demonstrate efficacy in restoration of motor function with a potency comparable to that seen with human fetal dopamine neurons. Furthermore, we show that hESC-derived dopamine neurons can project sufficiently long distances for use in humans, fully regenerate midbrain-to-forebrain projections, and innervate correct target structures. This provides strong preclinical support for clinical translation of hESC-derived dopamine neurons using approaches similar to those established with fetal cells for the treatment of Parkinson’s disease.

Parkinson's disease is typically treated with oral dopamine replacement therapies; however, long-term treatment leads to motor complications and, occasionally, impulse control disorders caused by intermittent stimulation of dopamine receptors and off-target effects, respectively. We aimed to assess the safety, tolerability, and efficacy of bilateral, intrastriatal delivery of ProSavin, a lentiviral vector-based gene therapy aimed at restoring local and continuous dopamine production in patients with advanced Parkinson's disease.

In order to perform in vivoimaging of the NR2B NMDA receptor system by positron emission tomography, a NR2B selective NMDA receptor antagonist has been labelled with carbon11 halflife: 20 min. N44fluorobenzylpiperidin1ylN′2oxo1,3dihydrobenzimidazol5yloxamide has been described demonstrating high affinity and selectivity for the NR2B receptors IC50of 5 nM in 3HRo25,6981 binding assay. The labelling precursor and the reference compound were synthesized by coupling the 44fluorobenzylpiperidine with the corresponding oxalamic acid. The reaction of 11Cphosgene with phenylenediamine precursor led the formation of the 11Cbenzimidazolone ring present on the ligand. The labelling occurred in THF or acetonitrile and the decay corrected radiochemical yield was 30–40 from the produced 11Cmethane. HPLC purification and formulation led to 2.6–3.7 GBq 70–100 mCi of radioligand within 30–35 min. The specific radioactivity was 72–127 GBqµmol 2–3.4 Ciµmol at the end of synthesis. Copyright © 2009 John Wiley & Sons, Ltd.

Recombinant lentiviral vectors (rLV) are powerful tools for gene transfer to the central nervous system (CNS) and hold great potential as a therapeutic gene therapy strategy for neurological disorders. Recent data indicate that rLVs are suitable for functional studies in the CNS by over expression or knock down of specific proteins. Based on a variety of lentiviruses species, different vector systems have been developed. However, the most commonly used rLV vector is based on the human immunodeficiency virus 1 (HIV-1). Here we describe the use of such vectors to achieve cell-specific transgene expression in the brain. In this setting, rLVs are versatile tools both due to their relatively large cloning capacity and their ability to transduce non-dividing cells. Furthermore, we discuss the preclinical development of gene therapy based on enzyme replacement and/or delivery of neurotrophic factors for neurodegenerative diseases and CNS manifestations of lysosomal storage diseases. Neuroprotective strategies that aim to deliver glial cell line-derived neurotrophic factor and ciliary neurotrophic factor for Parkinsons and Huntingtons diseases in particular have been documented with success in appropriate animal models. More recently, rLVs were shown to be suitable to express small interfering RNA for treatment in models of Alzheimers disease and amyotrophic lateral sclerosis. Finally, we present a review of the use of rLVs to model neurodegenerative diseases. rLVs have proven to be a very versatile tool to create genetic models of both Parkinsons and Huntingtons diseases and thus provide possibilities to study complex genetic interactions in otherwise wild-type animals evading the necessity to create transgenic mice. Moreover, the potential of these vectors in the development of gene therapy to treat neurological disorders is considerable, which is supported by the fact that clinical trials using rLVs are underway.

Recently, a new 2‐(iodophenyl)imidazo[1,2‐a]pyridineacetamide series has been developed as iodine‐123‐labelled radioligands for imaging the peripheral benzodiazepine receptors using single photon emission tomography. Within this series, 2‐[6‐chloro‐2‐(4‐iodophenyl)‐imidazo[1,2‐a]pyridin‐3‐yl]‐N‐ethyl‐N‐methyl‐acetamide (CLINME) was considered as an appropriate candidate for positron emission tomography imaging and was isotopically labelled with carbon‐11 (T1/2: 20.38 min) at the methylacetamide side chain from the corresponding nor‐analogue using [11C]methyl iodide and the following experimental conditions: (1) trapping at −10°C of [11C]methyl iodide in a 1/2 (v:v) mixture of DMSO/DMF (300 µl) containing 0.7–1.0 mg of the precursor for labelling and 3–5 mg of powdered potassium hydroxide (excess); (2) heating the reaction mixture at 110°C for 3 min under a nitrogen stream; (3) diluting the residue with 0.6 ml of the HPLC mobile phase; and (4) purification using semi‐preparative HPLC (Zorbax® SB18, Hewlett Packard, 250 × 9.4 mm). Typically, starting from a 1.5 Ci (55.5 GBq) [11C]CO2 production batch, 120−150 mCi (4.44–5.55 GBq) of [11C]CLINME were obtained (16–23% decay‐corrected radiochemical yield, n=12) within a total synthesis time of 24–27 min (Sep‐pak®Plus‐based formulation included). Specific radioactivities ranged from 0.9 to 2.7 Ci/µmol (33.3–99.9 GBq/µmol) at the end of radiosynthesis. Copyright © 2007 John Wiley & Sons, Ltd.

Alterations of mitochondrial function may play a central role in neuronal death in Huntington's disease (HD). However, the molecular mechanisms underlying such functional deficits of mitochondria are not elucidated yet. We herein showed that the expression of two important constituents of mitochondrial complex II, the 30-kDa iron-sulfur (Ip) subunit and the 70-kDa FAD (Fp) subunit, was preferentially decreased in the striatum of HD patients compared with controls. We also examined several mitochondrial proteins in striatal neurons that were infected with lentiviral vectors coding for the N-terminus part of huntingtin (Htt) with either a pathological (Htt171-82Q) or physiological (Htt171-19Q) polyglutamine tract. Compared with Htt171-19Q, expression of Htt171-82Q preferentially decreased the levels of Ip and Fp subunits and affected the dehydrogenase activity of the complex. The Htt171-82Q–induced preferential loss of complex II was not associated with a decrease in mRNA levels, suggesting the involvement of a posttranscriptional mechanism. Importantly, the overexpression of either Ip or Fp subunit restored complex II levels and blocked mitochondrial dysfunction and striatal cell death induced by Htt171-82Q in striatal neurons. The present results strongly suggest that complex II defects in HD may be instrumental in striatal cell death.

Abstract The transplantation of fetal porcine neurons is a potential therapeutic strategy for the treatment of human neurodegenerative disorders. A major obstacle to xenotransplantation, however, is the immune-mediated rejection that is resistant to conventional immunosuppression. To determine whether genetically modified donor pig neurons could be used to deliver immunosuppressive proteins locally in the brain, transgenic pigs were developed that express the human T cell inhibitory molecule hCTLA4-Ig under the control of the neuron-specific enolase promoter. Expression was found in various areas of the brain of transgenic pigs, including the mesencephalon, hippocampus and cortex. Neurons from 28-day old embryos secreted hCTLA4-Ig in vitro and this resulted in a 50% reduction of the proliferative response of human T lymphocytes in xenogenic proliferation assays. Transgenic embryonic neurons also secreted hCTLA4-Ig and had developed normally in vivo several weeks after transplantation into the striatum of immunosuppressed rats that were used here to study the engraftment in the absence of immunity. In conclusion, these data show that neurons from our transgenic pigs express hCTLA4-Ig in situ and support the use of this material in future pre-clinical trials in neuron xenotransplantation.

Huntington's disease (HD) is a monogenic neurodegenerative disease that affects the efferent neurons of the striatum. The protracted evolution of the pathology over 15 to 20 years, after clinical onset in adulthood, underscores the potential of therapeutic tools that would aim at protecting striatal neurons. Proteins with neuroprotective effects in the adult brain have been identified, among them ciliary neurotrophic factor (CNTF), which protected striatal neurons in animal models of HD. Accordingly, we have carried out a phase I study evaluating the safety of intracerebral administration of this protein in subjects with HD, using a device formed by a semipermeable membrane encapsulating a BHK cell line engineered to synthesize CNTF. Six subjects with stage 1 or 2 HD had one capsule implanted into the right lateral ventricle; the capsule was retrieved and exchanged for a new one every 6 months, over a total period of 2 years. No sign of CNTF-induced toxicity was observed; however, depression occurred in three subjects after removal of the last capsule, which may have correlated with the lack of any future therapeutic option. All retrieved capsules were intact but contained variable numbers of surviving cells, and CNTF release was low in 13 of 24 cases. Improvements in electrophysiological results were observed, and were correlated with capsules releasing the largest amount of CNTF. This phase I study shows the safety, feasibility, and tolerability of this gene therapy procedure. Heterogeneous cell survival, however, stresses the need for improving the technique.

Recent findings demonstrated the neuroprotective effects of insulin-like growth factor 1 (IGF-1) in a cultured cell model of Huntington's disease. In the present study, we examined the potential neuroprotective effect of IGF-1 in vivo, in a rat model of Huntington's disease using the NMDA receptor agonist quinolinate. Continuous intracerebroventricular infusion of recombinant IGF-1 (0.25 μgh for 2 days) in the rat brain, produced a significant 8-fold increase in striatal levels of the growth factor. Histological evaluation after intrastriatal injection of quinolinate showed that IGF-1 treatment significantly attenuated striatal degeneration. These results further support the therapeutic interest of IGF-1 in Huntington's disease.

Several neurotransmitter systems are involved in the pathogenesis of Huntington's disease. Here, we examined the involvement of cannabinoid CB1receptors in striatal degeneration in the rat model of this disease generated by administration of 3-nitropropionic acid (3NP). Several days before onset of striatal degeneration, G-protein activation by cannabinoid agonists was significantly decreased whereas density and mRNA levels of CB1receptors remained essentially normal. This change was transient, CB1receptors recovering full functionality after few days. Later, at onset of striatal degeneration, profound alterations of CB1receptors were detected, including marked reductions of their density, mRNA levels and coupling to G proteins. In these rats, the administration of the cannabinoid agonist Δ9-tetrahydrocannabinol was neuroprotective, which indicates that the early loss of CB1receptor signaling could be instrumental in 3NP toxicity. In conclusion, the present study supports the hypothesis that cannabinoid receptors, possibly the CB1receptor subtype, may be involved in HD pathogenesis and could be an interesting therapeutic target to slow disease progression.

Fetal neural transplants have become clinically relevant over the past 15 years for two major neurodegenerative diseases, namely Parkinson’s disease and Huntington’s disease. It is therefore timely to consider how this neurosurgical procedure can integrate the therapeutic armamentarium, what can be expected of it, and what cannot. We use here the example of Huntington’s disease to show what fetal neural transplants may uniquely offer for that disease. Up to very recent times, Huntington’s disease has been one special example of those neurodegenerative diseases against which neurologists feel totally helpless. This has all changed today and, although results are essentially still to come, one can foresee the mobilization of very large scientific and medical forces against this disease, with definite steps forward in terms of physiopathology and a better view of the therapeutic challenges. While defining the role that fetal neural transplantation may play in meeting these challenges, we also try to show rationales and developments for all types of treatments attempted or suggested so far, as well as their limits and, when relevant, informative failures. The date of writing this review needs to be noted, because the rapid accumulation of data on molecular mechanisms of Huntington’s disease pathogenesis and the increasing numbers of clinical trials do not allow much time for the ink of a review to dry.

Huntington’s disease is a hereditary disease in which degeneration of neurons in the striatum leads to motor and cognitive deficits. Foetal striatal allografts reverse these deficits in phenotypic models of Huntington’s disease developed in primates. A recent open‐label pilot study has shown some clinical improvement or stabilization in three out of five Huntington’s disease patients who received bilateral striatal grafts of foetal neurons. We show here that the clinical changes in these three patients were associated with a reduction of the striatal and cortical hypometabolism, demonstrating that grafts were able to restore the function of striato‐cortical loops. Conversely, in the two patients not improved by the grafts, striatal and cortical hypometabolism progressed over the 2‐year follow‐up. Finally, detailed anatomical–functional analysis of the grafted striata, enabled by the 3D fusion of MRI and metabolic images, revealed considerable heterogeneity in the anatomic and metabolic profiles of grafted tissue, both within and between Huntington’s disease patients. Our results demonstrate the usefulness of PET measurements of brain glucose metabolism in understanding the effects of foetal grafts in patients with Huntington’s disease.

Cannabinoids could provide neuroprotection in neurodegenerative disorders. In this study, we examined whether a treatment with 9-tetrahydrocannabinol, a non-selective cannabinoid receptor agonist, or with SR141716, a selective antagonist for the cannabinoid CB1receptor subtype, could affect the toxicity of the complex II reversible inhibitor malonate injected into the striatum, which replicates the mitochondrial complex II deficiency seen in Huntington's disease patients. As expected, malonate injection produced a significant reduction in cytochrome oxidase activity in the striatum consistent with the expected neurodegeneration caused by this toxin. The administration of 9-tetrahydrocannabinol increased malonate-induced striatal lesions compared to vehicle and, surprisingly, SR141716, far from producing effects opposite to those of 9-tetrahydrocannabinol, also enhanced malonate effects, and to an even greater extent. In summary, our results are compatible with the idea that manipulating the endocannabinoid system can modify neurodegeneration in Huntington's disease, and suggest that highly selective CB1receptor agonists might be necessary to produce neuroprotective effects against indirect excitotoxicity.

Assessment programs recently designed to follow‐up patients with Huntington's disease (HD) in therapeutic trials have not included electrophysiological testing in the list of mandatory examinations. This omission is likely due to the current lack of data establishing a clear correlation between the electrophysiological results and those of clinical assessment. We address this issue in a cohort of 36 patients at relatively early stages of the disease (I and II). Electrophysiological studies comprised the recording of palmar sympathetic skin responses (SSRs), blink reflexes (BRs), thenar long latency reflexes (LLRs), cortical somatosensory evoked potentials (SEPs), and electromyographic silent periods evoked by transcranial magnetic stimulation (SPs). Results were analyzed with reference to disease duration and staging and to specific cognitive, psychiatric, and motor alteration. SEPs were the most and very sensitive markers, because they were abnormal in 94% of patients. Except for LLRs, alteration of electrophysiological results increased in parallel to the evolution of the disease. Except for LLRs and SSR latency, electrophysiological results correlated with those of specific clinical examinations. In particular, an increased BR latency or a reduced amplitude of the N20 component of SEPs correlated with the extent of bradykinesia, whereas a reduced amplitude of SSRs or of the N30 component of SEPs correlated with hyperkinesia. Overall, electrophysiological tests, in particular SEPs and BRs, appeared sensitive and interesting in the follow‐up of HD patients and correlated with various clinical parameters, suggesting that these easy to perform and noninvasive repeatable examinations could be added fruitfully to the assessment programs for HD. © 2002 Movement Disorder Society

Intrastriatal transplantation of striatal neuroblasts from human fetuses is a promising approach for treatment of Huntington's disease, on the basis of many experimental animal studies and, most recently, pilot clinical trials. Technically, several issues remain to be resolved (e.g., the precise site of dissection of the fetal tissue; the number and location of the fetal striatal implants; or the use of immunosuppressive therapy), and await larger‐scale trials and purposely designed protocols. Further clinical data must also be obtained, and preliminary promising results must be replicated in a patient group large enough to provide conclusive results. It is important to establish (1) the amount of clinical benefit provided to the patient by the grafted cells; (2) the anticipated duration of clinical benefits; and (3) the secondary rate of decline after the benefit of the graft has been overbalanced. Evaluation of these parameters will require very long‐term follow‐up of the patients involved, over several years after grafting, before the technique can eventually be proposed widely to patients. © 2002 Movement Disorder Society

Huntington's disease (HD) is generally considered a prototypic motor disorder, but cognitive deficits are also prominent features of the disease. Systemic administration of the mitochondrial toxin 3-nitropropionic acid (3NP) has been proposed to be a phenotypic model of HD in rats and nonhuman primates. In this study, we investigated the effect of 5 days continuous subcutaneous infusion of 3NP on motor and cognitive abilities in Lewis rats. Intoxicated animals developed a motor syndrome consisting of bradykinesia as well as gait abnormalities and dystonic hindlimbs. Results from learning tasks showed that these rats: (1) did not exhibit learning deficits per se in our discrimination task but showed impairments in inhibiting behavioral responses when a transfer of learning (to new stimuli) or a transfer of response (new position of the lever) was required; (2) showed a marked tendency to persevere in choosing the compartment they previously visited in a T maze, thus leading to a clear retardation in learning a reinforced alternation task; and (3) did not show any memory deficit when a delay was introduced. Six months later, histological analyses showed severe neurodegeneration within the lateral striatum accompanied by apparent cell loss in the ventral pallidum and entopedoncular nucleus. We suggest that the 3NP rat model of basal ganglia neurodegeneration may provide a useful model for studying certain fundamental aspects of the physiopathology of HD and for evaluating the functional efficacy of new therapeutic strategies.

Positron emission tomography (PET) coupled to 6-[18F]Fluoro-L-Dopa (18F-Dopa) remains the gold standard for assessing dysfunctionality concerning the dopaminergic nigrostriatal pathway in Parkinson's disease and related disorders. The use of ligands of the dopamine transporters (DAT) is an attractive alternative target; consequently, the current aim was to validate one of them, 11C-PE2I, using a multiinjection modeling approach allowing accurate quantitation of DAT densities in the striatum. Experiments were performed in three controls, three MPTP-treated (parkinsonian) baboons, and one reserpine-treated baboon. 11C-PE2I B′max values obtained with this approach were compared with 18F-Dopa input rate constant values (Ki), in vitroBmax binding of 125I-PE2I, and the number of dopaminergic neurons in the substantia nigra estimated postmortem by stereology. In the caudate nucleus and putamen, control values for 11C-PE2I B'max were 673 and 658 pmol/mL, respectively, whereas it was strongly reduced in the MPTP-treated (B′max = 26 and 36 pmol/mL) and reserpine-treated animals (B′max = 338 and 483 pmol/mL). In vivo11C-PE2I B′max values correlated with 18F-Dopa Ki values and in vitro125I-PE2I Bmax values in the striatum and with the number of nigral dopaminergic neurons. Altogether, these data support the use of 11C-PE2I for monitoring striatal dopaminergic disorders and the effect of potential neuroprotective strategies.

A new scheme is proposed to edit the 3.0 ppm GABA resonance without macromolecule (MM) contamination. Like previous difference spectroscopy approaches, the new scheme manipulates J‐modulation of this signal using a selective editing pulse. The elimination of undesirable MM contribution at 3.0 ppm is obtained by applying this pulse symmetrically about the J‐coupled MM resonance, at 1.7 ppm, in the two steps of the editing scheme. The effectiveness of the method is demonstrated in vitro, using lysine to mimic MM, and in vivo. As compared to the most commonly used editing scheme, which necessitates the acquisition and processing of two distinct difference spectroscopy experiments, the new scheme offers a reduction in experimental time (–33%) and an increase in accuracy. Magn Reson Med 45:517–520, 2001. © 2001 Wiley‐Liss, Inc.

Huntington's disease (HD) is an inherited disorder characterized by cognitive impairments, motor deficits, and progressive dementia. These symptoms result from progressive neurodegenerative changes mainly affecting the neostriatum. This pathology is fatal in 10 to 20 years and there is currently no treatment for HD. Early in the course of the disease, initial clinical manifestations are due to striatal neuronal dysfunction, which is later followed by massive neuronal death. A major therapeutic objective is therefore to reverse striatal dysfunction prior to cell death. Using a primate model reproducing the clinical features and the progressive neuronal degeneration typical of HD, we tested the therapeutic effects of direct intrastriatal infusion of ciliary neurotrophic factor (CNTF). To achieve a continuous delivery of CNTF over the full period of evaluation, we took advantage of the macroencapsulation technique. Baby hamster kidney (BHK) cells previously engineered to produce human CNTF were encapsulated into semipermeable membranes and implanted bilaterally into striata. We show here that intracerebral delivery of low doses of CNTF at the onset of symptoms not only protects neurons from degeneration but also restores neostriatal functions. CNTF-treated primates recovered, in particular, cognitive and motor functions dependent on the anatomofunctional integrity of frontostriatal pathways that were distinctively altered in this HD model. These results support the hypothesis that CNTF infusion into the striatum of HD patients not only could block the degeneration of neurons but also alleviated motor and cognitive symptoms associated with persistent neuronal dysfunction.

N-acetylaspartate (NAA) quantification by 1H-magnetic resonance spectroscopy has been commonly used to assess in vivoneuronal loss in neurodegenerative disorders. Here, the authors used ex vivoand in vivo1H-magnetic resonance spectroscopy in rat and primate models of progressive striatal degeneration induced by the mitochondrial toxin 3-nitropropionate (3NP) to determine whether early NAA depletions could also be associated with neuronal dysfunction. In rats that were treated for 3 days with 3NP and had motor symptoms, the authors found a significant decrease in NAA concentrations, specifically restricted to the striatum. No cell loss or dying cells were found at this stage in these animals. After 5 days of 3NP treatment, a further decrease in striatal NAA concentrations was observed in association with the occurrence of dying neurons in the dorsolateral striatum. In 3NP-treated primates, a similar striatal-selective and early decrease in NAA concentrations was observed after only a few weeks of neurotoxic treatment, without any sign of ongoing cell death. This early decrease in striatal NAA was partially reversed after 4 weeks of 3NP withdrawal. These results demonstrate that early NAA depletions reflect a reversible state of neuronal dysfunction preceding cell degeneration and suggest that in vivoquantification of NAA 1H-magnetic resonance spectroscopy may become a valuable tool for assessing early neuronal dysfunction and the effects of potential neuroprotective therapies in neurodegenerative disorders.

Delayed abnormal movements can be observed in patients with acute neurologic insult after a prolonged period of apparent neurologic stability. To reproduce such a secondary neurologic manifestation in primates, the present experiment investigated whether systemic administration of subacute 3‐nitropropionic acid (3NP), a mitochondrial toxin, could induce abnormal movements that were delayed and progressive over time. Four Cebus apellamonkeys received systemic 3NP injections until acute neurologic signs manifested. The monkeys were regularly video‐recorded and rated for abnormal movements for up to 15 weeks after the cessation of 3NP treatment. Five to 6 weeks after the 3NP treatment, monkeys displayed a significant increase in dyskinesias compared with pretreatment conditions. Over time the chorea attenuated, whereas the dystonic movements increased in intensity and severity which was characterized by a delayed decrease of peak tangential velocity. The intensity of abnormal movements and extent of affected body regions observed in each monkey were consistent with the size of basal ganglia hypersignal as documented by T2 sequence on magnetic resonance imaging. Thus, more severe motor impairments were associated with large magnetic resonance image abnormalities. This novel primate model may be particularly useful for studying the structural changes underlying delayed and progressive manifestations of abnormal movements with the ultimate goal of facilitating the evaluation of novel therapeutic strategies.

This study describes issues related to the safety and tolerability of fetal striatal neural allografts as assessed in five patients with Huntington's disease. Huntington's disease (HD) is characterized by motor, cognitive, and behavioral disturbances. The latter include psychological disturbances and, as a consequence, we took particular care to analyze behavioral changes, in addition to the usual “safety” follow-up. We conducted multidisciplinary follow-up at least 2 years before and 1 year after grafting. Psychological care extended to close relatives. The grafting procedure itself was altogether safe and uneventful, and there were no apparent clinical deleterious effects for 1 year. The immunosuppressive treatment, however, was complicated by various problems (irregular compliance, errors of handling, side effects). Direct psychological consequences of the transplantation procedure were rare and not worrisome, although mood alteration requiring treatment was observed in one patient. Indirectly, however, the procedure required patients and relatives to accept constraints that tended to complicate familial situations already marred by aggressivity and depression. All patients and close relatives expressed major expectations, in spite of our strong and repeated cautioning. It is clearly important to be aware of these particular conditions since they may eventually translate into psychological difficulties in coping with the long-term clinical outcome of the procedure, if not beneficial. Despite an overall good tolerance, therefore, this follow-up calls for caution regarding the involvement of HD patients in experimental surgical protocols.

Previous studies in primates have shown that chronic systemic administration of the succinate dehydrogenase (SDH) inhibitor, 3-nitropropionic acid (3NP), replicates most of the motor, cognitive, and histopathological features of Huntington's disease. In the present study, serial 1H-NMR spectroscopy (1H-MRS) assessment of striatal and occipital cortex concentrations of N-acetylaspartate, phosphocreatine/creatine, choline, and lactate, were obtained every 2-weeks during the entire course of a chronic 3NP treatment in baboons. A region-selective increase in lactate was detected in the striatum of the 3NP-treated animals, either immediately before or in conjunction with a lesion in the dorsolateral putamen detected by T2-MR imaging. Absolute 1H-MRS quantitation demonstrated a progressive and region-specific decrease in striatal N-acetylaspartate, creatine, and choline, occuring as early as 3 weeks before the first detection of lactate. These results demonstrate that 1H-MRS can be used to monitor early stages of brain metabolic impairment. In addition, given that 3NP-induced SDH inhibition following systemic injection similarly affects all brain regions, the striatal selective decreases in N-acetylaspartate or creatine concentrations are not simply related to the level of mitochondrial impairment but to a preferential vulnerability of the striatum to 3NP-induced toxicity.

Lentiviral vectors infect quiescent cells and allow for the delivery of genes to discrete brain regions. The present study assessed whether stable lentiviral gene transduction can be achieved in the monkey nigrostriatal system. Three young adult Rhesus monkeys received injections of a lentiviral vector encoding for the marker gene β galatosidase (βGal). On one side of the brain, each monkey received multiple lentivirus injections into the caudate and putamen. On the opposite side, each animal received a single injection aimed at the substantia nigra. The first two monkeys were sacrificed 1 month postinjection, while the third monkey was sacrificed 3 months postinjection. Robust incorporation of the βGal gene was seen in the striatum of all three monkeys. Stereological counts revealed that 930,218; 1,192,359; and 1,501,217 cells in the striatum were βGal positive in monkeys 1 (n=2) and 3 (n=1) months later, respectively. Only the third monkey had an injection placed directly into the substantia nigra and 187,308 βGal-positive cells were identified in this animal. The injections induced only minor perivascular cuffing and there was no apparent inflammatory response resulting from the lentivirus injections. Double label experiments revealed that between 80 and 87% of the βGal-positive cells were neurons. These data indicate that robust transduction of striatal and nigral cells can occur in the nonhuman primate brain for up to 3 months. Studies are now ongoing testing the ability of lentivirus encoding for dopaminergic trophic factors to augment the nigrostriatal system in nonhuman primate models of Parkinson's disease.

In this article, we show that 1) computed tomographic (CT)-guided stereotactic infusion of an excitotoxin into the striatum of a nonhuman primate provides a useful neuropathologic and behavioral model for Huntington's disease. 2) High-resolution positron emission tomography (PET) can be used to image the decreased glucose utilization and the preservation of dopaminergic terminals in the lesioned striatum by using 2-fluoro-deoxy-D-glucose (2FDG) and N-(C-11)-methyl-2-beta-carbomethoxy-3-beta-phenyl tropane (CPT) as tracers. 3) Transplantation of crossspecies striatal fetal tissue into the lesioned caudate-putamen reduces many of the abnormal motor movements and behavioral changes seen in the Huntington's disease primate model. 4) Graft rejection results in the return of the abnormal signs of the pregrafted state. These results indicate that treatment of the neuronal deficit in Huntington's disease can involve intervention at the local neuronal circuit level. CT-guided stereotactic implantation of cells that might protect or replace this defective circuitry may eventually provide an effective treatment for Huntington's disease.

In vivo benzodiazepine receptor occupancy by increasing doses of CL 218,872 has been evaluated in the baboon Papio papio, using (11C) RO 15-1788 as specific radioligand and positron emission tomography as external detection system. Although BZR heterogeneity has been previously demonstrated in the brain of the living baboon using PET, we did not observe in our studies that CL 218,872 interacts preferentially with one of the BZR subtypes. The monophasic pattern of the dose dependent CL 218,872 displacement curve and the corresponding “in vivo Hill coefficient” near unity suggest that CL 218,872 binds in cerebral baboon cortex with a similar affinity with BZ1 as well as BZ2 subtypes. The anticon-vulsant properties of CL 218,872 against bicuculline and allylglycine-induced seizures were correlated with benzodiazepine receptor occupancy by assessment of electroencephalographic activity during positron emission tomography studies. Our data confirmed in vivo the hypothesis of a partial agonist anticonvulsant activity of CL 218,872. At the same time, the use of a GABAantagonist (bicuculline) or an inhibitor of the GABA synthesis (allylglycine) suggested the existence of an allosteric interaction between benzodiazepine receptors and GABA receptors.

Compelling evidence suggests that a defect in energy metabolism may play a role in the pathogenesis of various degenerative disorders including Parkinson's disease and Huntington's disease. The behavioural and neuropathological consequences in primates of chronic systemic administration of mitochondrial toxins which impair oxidative metabolism are of considerable interest. In the past few years observations have been published describing the behavioural, biochemical and histological consequences of chronic low-dose systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or 3-nitropropionic acid in baboons. The results of these studies strongly support the view that chronic energy metabolism impairment is involved in the pathogenesis of Parkinson's and Huntington's diseases.

Ibotenic acid was injected unilaterally into the baboon caudate-putamen (CP) to achieve a neural degeneration model in the primate, with a neuropathology similar to Huntington's disease. Four to six weeks later injections of cell suspensions of striatal precursor cells, obtained by dissection of the fetal rat striatal region (13–15 days gestational age), were made into the excitotoxically lesioned CP of 3 baboons immunosuppressed by Cyclosporin A. Morphological analysis indicated that in one of the baboons, which had the largest lesion of the CP and the shortest survival time (6 weeks after implantation), there was a surviving striatal implant. The implanted neurons grew in high densities in cellular aggregates within the host gliotic CP. These neurons had a neuronal size phenotypical for rat striatum, i.e. on average about a 25% smaller neuronal cell diameter than a similar population in the baboon caudate-putamen. Glial-fibrillary-acid-protein immunoreactivity was present on large astrocytes within the striatal implant, with a distinct border towards the lesion-induced astrogliosis of the host. Neuronal markers for acetylcholinesterase and Leu-enkephalin were distributed in a typical patchy manner in the striatal implants along with fiber staining for tyrosine-hydroxylase-like immunoreactivity (TH) possibly derived from afferent host dopaminergic axons. Some of these fibers in the implants came from intrinsic TH-positive neuronal somata, probably of neocortical fetal origin and transiently expressing the enzyme. In conclusion, the results indicate that neuronal replacement can be achieved by crossspecies implantation of fetal striatal precursor cells to the previously neuron depleted primate CP under immunosuppression but that the survival and growth of such implants may be variable and subject to unfavourable trophic conditions.

Substitutive therapy using fetal striatal grafts in animal models of Huntington disease (HD) have already demonstrated obvious beneficial effects on motor indices1. Using a new phenotypic model of HD recently designed in primates2,3, we demonstrate here complete and persistent recovery in a frontal-type cognitive task two to five months after intrastriatal allografting. The striatal allografts also reduce the occurence of dystonia, a major abnormal movement associated with HD. These results show the capacity of fetal neurons to provide a renewed substrate for both cognitive and motor systems in the lesioned adult brain. They also support the use of neural transplantation as a potential therapy for HD.

MONKEYS were treated on two regimens of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injections to achieve dopamine fiber degeneration of differing severities. A rapid treatment regimen produced a severe parkinsonian syndrome, whereas an intermittent regimen did not cause locomotor symptoms to appear up to 25 weeks. High resolution PET scanning of dopamine nerve terminals revealed that the specific binding of the dopamine transporter [11C]-WIN 35,428 ([11C]-CFT) was diminished by 94 (caudate nucleus) and by 93 (putamen) in the symptomatic monkey. Decreases of 65 and 67 were detected in these regions in the non-symptomatic monkey. Post-mortem immunocytochemical evaluation of presumed dopamine fibers by tyrosine-hydroxylase showed similar reductions in the symptomatic animal.

In vivophysiological changes associated with striatal pathology were determined by measurement of glucose utilization, binding to D1receptors and dopamine reuptake sites, regional blood flow, and behavior before and after unilateral quinolinate infusions into caudate-putamen in three nonhuman primates (Macaca fascicularisand Macaca mulata). Following the quinolinate lesion, symptoms similar to those of Huntington's disease could be induced by dopamine agonist treatment. In addition, all animals showed a long-term decrease in glucose utilization in the caudate by [18F]fluoro-2-deoxy-d-glucose positron emission tomography (PET). At 4-6 weeks following the lesion the average decrease in glucose utilization in the caudate-putamen was between 40 and 50% of the prelesion values in primates with large lesions. Corresponding caudate-putamen regional blood volume in these animals showed a 61 and 74% decrease as studied by magnetic resonance imaging with somewhat smaller changes observed in an index of cerebral blood flow. The caudate-putamen uptake rate constants for D1receptors reflected neuronal loss and decreased by an average 40 and 48%, as determined by 11C-labeled Schering compound (SCH 39 166) and PET. Dopamine reuptake sites and fibers assessed by the 11C-labeled cocaine analog, WIN 35 428 compound, and PET showed a temporary decrease in areas with mild neuronal loss and a long-term decrease in striatal regions with severe destruction. These results, which were consistent with behavioral changes and neuropathology seen at postmortem examination, can be related to in vivophysiological studies of Huntington's disease patients.

Optimization of the procedures for neural grafting is a timely issue, as this technique has proven beneficial for a few patients with late-stage Parkinson's disease in pilot studies and therefore may expand to become a more widely available therapeutic. In this research, one major issue is that of the placement of the cell deposits in the right target areas within the striatum. Although it is widely accepted that these suitable regions are the sensorimotor regions of the putamen, reliable delineation of these areas using classical stereotactical mapping techniques remains difficult. Along the course of a 5-year-long clinical transplantation program, we have developed an original procedure based on magnetic resonance imaging of the striatum on parasagittal views. This technique allowed us to identify precisely, and reproducibly in each patient, three subregions of the putamen (precommissural, commissural, and postcommissural) to be implanted. On the basis of the literature defining the sensorimotor putaminal regions in nonhuman primates, it was subsequently possible to extrapolate and localize these regions in each patient, thus providing a basis for the placement of cell deposits. Examples taken from our series of grafted patients demonstrate the value of this procedure that, in addition, minimizes interference of interindividual variability in the interpretation of clinical results.

After a number of reports indicating positive clinical outcome of intrastriatal transplantation of fetal ventral mesencephalic tissue into patients with Parkinson’s disease, the time may have come to consider the possibility of using this technique to treat patients with Huntington’s disease. On the basis of the available literature, the Network of European CNS Transplantation and Restoration has established a program aiming at defining the optimal conditions for such clinical trials. The present study, conducted within this framework, pursued the goal of providing information concerning the period of striatal neuronal ontogeny in humans, taking into account the technical and legal requirements imposed by the clinical procedure of neural transplantation using human tissue. On this basis, it aimed at establishing a reliable dissecting method for the intrastriatal grafting of human fetal striatal neurons. The ontogeny of medium-spiny neurons within the developing striatum was first studied in a series of human fetal brains, 5 to 10 weeks postconception, using immunocytochemical detection of DARPP-32. Immunoreactive neurons were observed in fetuses at 7 weeks of age and older. They were mostly localized in clusters, packed in the lateral ganglionic eminence. Over a 2-week-long period, DARPP-32 neurons increased in number. Their morphology remained poorly differentiated, however, with small cell bodies, few branched dendrites, and variable intensity of immunostaining. Based on these findings, selective dissection of the lateral ganglionic eminence was carried out. This tissue was stereotaxically implanted into the striatum of immunosuppressed adult rats previously lesioned. Two months postgrafting, DARPP-32 neurons were observed as discrete patches, embedded within areas of essentially DARPP-32-negative tissue. Up to 2 months after grafting, neurons remained poorly differentiated in general, with only a few neurons exhibiting a dense immunoreactivity and long processes. These results indicate that striatal DARPP-32-immunoreactive neurons are present in the lateral ganglionic eminence in fetuses as soon as 7 weeks postconception. The striatal tissue can be dissected out and successfully transplanted. Within the grafts, neuronal differentiation appears to be a very long process, suggesting that many months might be necessary for these neurons to become functionally integrated into an adult host brain.

Riluzole has been shown recently to increase life expectancy in patients with amyotrophic lateral sclerosis. A number of experimental studies also suggest that this compound may be a neuroprotectant. We have investigated in baboons whether riluzole would protect striatal neurons from a prolonged 3-nitropropionic acid (3NP) treatment and ameliorate the associated motor symptoms. In animals receiving 3NP and the solvent of riluzole, 12 weeks of high-dose 3NP treatment resulted in the appearance of persistent leg dystonia and significant increases in the incidence of three categories of abnormal movements and in the dyskinesia index in the apomorphine test (0.5 mg/kg im). Quantitative assessment of these behavioral deficits using a video movement analysis system demonstrated a significant decrease in locomotor activity and peak tangential velocity in 3NP-treated animals compared to controls. Histological analysis showed the presence of severe, bilateral, striatal lesions, localized in both caudate and putamen. Cotreatment with riluzole (4 mg/kg ip, twice daily) significantly reduced the dyskinesia index (−35%,P<0.02) in the apomorphine test. In the quantitative behavioral analysis, riluzole significantly ameliorated the decrease in peak tangential velocity (P<0.02) but not the decrease in locomotor activity observed after 3NP. Comparative histological analysis of the two groups of treated animals did not demonstrate a clear neuroprotective effect of riluzole. The present study suggests that one potential therapeutic interest for riluzole in neurodegenerative disorders may reside in the reduction of motor symptoms associated with striatal lesions.