An Update on Gene Therapy Approaches for Parkinson's Disease: Restoration of Dopaminergic Function.

At present there is a significant unmet need for clinically available treatments for Parkinson's disease (PD) patients to stably restore balance to dopamine network function, leaving patients with inadequate management of symptoms as the disease progresses. Gene therapy is an attractive approach to impart a durable effect on neuronal function through introduction of genetic material to reestablish dopamine levels and/or functionally recover dopaminergic signaling by improving neuronal health. Ongoing clinical gene therapy trials in PD are focused on enzymatic enhancement of dopamine production and/or the restoration of the nigrostriatal pathway to improve dopaminergic network function. In this review, we discuss data from current gene therapy trials for PD and recent advances in study design and surgical approaches.

Gene therapy for aromatic L-amino acid decarboxylase deficiency by MR-guided direct delivery of AAV2-AADC to midbrain dopaminergic neurons.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare genetic disorder characterized by deficient synthesis of dopamine and serotonin. It presents in early infancy, and causes severe developmental disability and lifelong motor, behavioral, and autonomic symptoms including oculogyric crises (OGC), sleep disorder, and mood disturbance. We investigated the safety and efficacy of delivery of a viral vector expressing AADC (AAV2-hAADC) to the midbrain in children with AADC deficiency (ClinicalTrials.gov Identifier NCT02852213). Seven (7) children, aged 4-9 years underwent convection-enhanced delivery (CED) of AAV2-hAADC to the bilateral substantia nigra (SN) and ventral tegmental area (VTA) (total infusion volume: 80 µL per hemisphere) in 2 dose cohorts: 1.3 × 1011 vg (n = 3), and 4.2 × 1011 vg (n = 4). Primary aims were to demonstrate the safety of the procedure and document biomarker evidence of restoration of brain AADC activity. Secondary aims were to assess clinical improvement in symptoms and motor function. Direct bilateral infusion of AAV2-hAADC was safe, well-tolerated and achieved target coverage of 98% and 70% of the SN and VTA, respectively. Dopamine metabolism was increased in all subjects and FDOPA uptake was enhanced within the midbrain and the striatum. OGC resolved completely in 6 of 7 subjects by Month 3 post-surgery. Twelve (12) months after surgery, 6/7 subjects gained normal head control and 4/7 could sit independently. At 18 months, 2 subjects could walk with 2-hand support. Both the primary and secondary endpoints of the study were met. Midbrain gene delivery in children with AADC deficiency is feasible and safe, and leads to clinical improvements in symptoms and motor function.

Cerebellomedullary Cistern Injection of Viral Vectors in Nonhuman Primates.

Gene therapy shows great promise for the treatment of neurological disorders, and accessing cerebrospinal fluid (CSF) from the cerebellomedullary cistern through the posterior atlanto-occipital membrane has become a common route of delivery in preclinical studies. Unlike direct brain parenchymal infusions, CSF delivery offers broader coverage to the central and peripheral nervous system. This prospectively increases its translational value, more specially to treat global brain dysfunctions in which the pathology is disseminated throughout the brain and not focalized in one specific brain structure. Also, from the practical point of view, this approach offers a more reliable method for neurological gene replacement in infants, whose immature cranial suture preclude the use of skull-mounted devices. Here we describe a consistent, precise, and safe method for CSF injection.

Kinetics and MR-Based Monitoring of AAV9 Vector Delivery into Cerebrospinal Fluid of Nonhuman Primates.

Here we evaluated the utility of MRI to monitor intrathecal infusions in nonhuman primates. Adeno-associated virus (AAV) spiked with gadoteridol, a gadolinium-based MRI contrast agent, enabled real-time visualization of infusions delivered either via cerebromedullary cistern, lumbar, cerebromedullary and lumbar, or intracerebroventricular infusion. The kinetics of vector clearance from the cerebrospinal fluid (CSF) were analyzed. Our results highlight the value of MRI in optimizing the delivery of infusate into CSF. In particular, MRI revealed differential patterns of infusate distribution depending on the route of delivery. Gadoteridol coverage analysis showed that cerebellomedullary cistern delivery was a reliable and effective route of injection, achieving broad infusate distribution in the brain and spinal cord, and was even greater when combined with lumbar injection. In contrast, intracerebroventricular injection resulted in strong cortical coverage but little spinal distribution. Lumbar injection alone led to the distribution of MRI contrast agent mainly in the spinal cord with little cortical coverage, but this delivery route was unreliable. Similarly, vector clearance analysis showed differences between different routes of delivery. Overall, our data support the value of monitoring CSF injections to dissect different patterns of gadoteridol distribution based on the route of intrathecal administration.

Extensive Transduction and Enhanced Spread of a Modified AAV2 Capsid in the Non-human Primate CNS.

The present study was designed to characterize transduction of non-human primate brain and spinal cord with a modified adeno-associated virus serotype 2, incapable of binding to the heparan sulfate proteoglycan receptor, referred to as AAV2-HBKO. AAV2-HBKO was infused into the thalamus, intracerebroventricularly or via a combination of both intracerebroventricular and thalamic delivery. Thalamic injection of this modified vector encoding GFP resulted in widespread CNS transduction that included neurons in deep cortical layers, deep cerebellar nuclei, several subcortical regions, and motor neuron transduction in the spinal cord indicative of robust bidirectional axonal transport. Intracerebroventricular delivery similarly resulted in widespread cortical transduction, with one striking distinction that oligodendrocytes within superficial layers of the cortex were the primary cell type transduced. Robust motor neuron transduction was also observed in all levels of the spinal cord. The combination of thalamic and intracerebroventricular delivery resulted in transduction of oligodendrocytes in superficial cortical layers and neurons in deeper cortical layers. Several subcortical regions were also transduced. Our data demonstrate that AAV2-HBKO is a powerful vector for the potential treatment of a wide number of neurological disorders, and highlight that delivery route can significantly impact cellular tropism and pattern of CNS transduction.

Depletion of AADC activity in caudate nucleus and putamen of Parkinson's disease patients; implications for ongoing AAV2-AADC gene therapy trial.

In Parkinson's disease (PD), aromatic L-amino acid decarboxylase (AADC) is the rate-limiting enzyme in the conversion of L-DOPA (Sinemet) to dopamine (DA). Previous studies in PD animal models demonstrated that lesion of dopaminergic neurons is associated with profound loss of AADC activity in the striatum, blocking efficient conversion of L-DOPA to DA. Relatively few studies have directly analyzed AADC in PD brains. Thus, the aim of this study was to gain a better understanding of regional changes in AADC activity, DA, serotonin and their monoamine metabolites in the striatum of PD patients and experimentally lesioned animals (rat and MPTP-treated nonhuman primate, NHP). Striatal AADC activity was determined post mortem in neuropathologically confirmed PD subjects, animal models and controls. A regional analysis was performed for striatal AADC activity and monoamine levels in NHP tissue. Interestingly, analysis of putaminal AADC activity revealed that control human striatum contained much less AADC activity than rat and NHP striata. Moreover, a dramatic loss of AADC activity in PD striatum compared to controls was detected. In MPTP-treated NHP, caudate nucleus was almost as greatly affected as putamen, although mean DA turnover was higher in caudate nucleus. Similarly, DA and DA metabolites were dramatically reduced in different regions of PD brains, including caudate nucleus, whereas serotonin was relatively spared. After L-DOPA administration in MPTP-treated NHP, very poor conversion to DA was detected, suggesting that AADC in NHP nigrostriatal fibers is mainly responsible for L-DOPA to DA conversion. These data support further the rationale behind viral gene therapy with AAV2-hAADC to restore AADC levels in putamen and suggest further the advisability of expanding vector delivery to include coverage of anterior putamen and the caudate nucleus.

Validation of antibodies for neuroanatomical localization of the P2Y11 receptor in macaque brain.

Focus on the purinergic receptor P2Y11 has increased following the finding of an association between the sleep disorder narcolepsy and a genetic variant in P2RY11 causing decreased gene expression. Narcolepsy is believed to arise from an autoimmune destruction of the hypothalamic neurons that produce the neuropeptide hypocretin/orexin. It is unknown how a decrease in expression of P2Y11 might contribute to an autoimmune reaction towards the hypocretin neurons and the development of narcolepsy. To advance narcolepsy research it is therefore extremely important to determine the neuroanatomical localization of P2Y11 in the brain with particular emphasis on the hypocretin neurons. In this article we used western blot, staining of blood smears, and flow cytometry to select two antibodies for immunohistochemical staining of macaque monkey brain. Staining was seen in neuron-like structures in cortical and hypothalamic regions. Rats do not have a gene orthologue to the P2Y11 receptor and therefore rat brain was used as negative control tissue. The chromogenic signal observed in macaque monkey brain in neurons was not considered reliable, because the antibodies stained rat brain in a similar distribution pattern. Hence, the neuroanatomical localization of the P2Y11 receptor remains undetermined due to the lack of specific P2Y11 antibodies for brain immunohistochemistry.

Widespread AAV1- and AAV2-mediated transgene expression in the nonhuman primate brain: implications for Huntington's disease.

Huntington's disease (HD) is caused by a toxic gain-of-function associated with the expression of the mutant huntingtin (htt) protein. Therefore, the use of RNA interference to inhibit Htt expression could represent a disease-modifying therapy. The potential of two recombinant adeno-associated viral vectors (AAV), AAV1 and AAV2, to transduce the cortico-striatal tissues that are predominantly affected in HD was explored. Green fluorescent protein was used as a reporter in each vector to show that both serotypes were broadly distributed in medium spiny neurons in the striatum and cortico-striatal neurons after infusion into the putamen and caudate nucleus of nonhuman primates (NHP), with AAV1-directed expression being slightly more robust than AAV2-driven expression. This study suggests that both serotypes are capable of targeting neurons that degenerate in HD, and it sets the stage for the advanced preclinical evaluation of an RNAi-based therapy for this disease.

AAV viral vector delivery to the brain by shape-conforming MR-guided infusions.

Gene transfer technology offers great promise as a potential therapeutic approach to the brain but has to be viewed as a very complex technology. Success of ongoing clinical gene therapy trials depends on many factors such as selection of the correct genetic and anatomical target in the brain. In addition, selection of the viral vector capable of transfer of therapeutic gene into target cells, along with long-term expression that avoids immunotoxicity has to be established. As with any drug development strategy, delivery of gene therapy has to be consistent and predictable in each study subject. Failed drug and vector delivery will lead to failed clinical trials. In this article, we describe our experience with AAV viral vector delivery system, that allows us to optimize and monitor in real time viral vector administration into affected regions of the brain. In addition to discussing MRI-guided technology for administration of AAV vectors we have developed and now employ in current clinical trials, we also describe ways in which infusion cannula design and stereotactic trajectory may be used to maximize the anatomical coverage by using fluid backflow. This innovative approach enables more precise coverage by fitting the shape of the infusion to the shape of the anatomical target.

SAFETY AND TOLERABILITY OF MRI-GUIDED INFUSION OF AAV2-hAADC INTO THE MID-BRAIN OF NON-HUMAN PRIMATE.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare, autosomal-recessive neurological disorder caused by mutations in the DDC gene that leads to an inability to synthesize catecholamines and serotonin. As a result, patients suffer compromised development, particularly in motor function. A recent gene replacement clinical trial explored putaminal delivery of recombinant adeno-associated virus serotype 2 vector encoding human AADC (AAV2-hAADC) in AADC-deficient children. Unfortunately, patients presented only modest amelioration of motor symptoms, which authors acknowledged could be due to insufficient transduction of putamen. We hypothesize that, with the development of a highly accurate MRI-guided cannula placement technology, a more effective approach might be to target the affected mid-brain neurons directly. Transduction of AADC-deficient dopaminergic neurons in the substantia nigra and ventral tegmental area with locally infused AAV2-hAADC would be expected to lead to restoration of normal dopamine levels in affected children. The objective of this study was to assess the long-term safety and tolerability of bilateral AAV2-hAADC MRI-guided pressurized infusion into the mid-brain of non-human primates. Animals received either vehicle, low or high AAV2-hAADC vector dose and were euthanized 1, 3 or 9 months after surgery. Our data indicate that effective mid-brain transduction was achieved without untoward effects.

Translational fidelity of intrathecal delivery of self-complementary AAV9-survival motor neuron 1 for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations in survival motor neuron 1 (SMN1). Previously, we showed that central nervous system (CNS) delivery of an adeno-associated viral (AAV) vector encoding SMN1 produced significant improvements in survival in a mouse model of SMA. Here, we performed a dose-response study in SMA mice to determine the levels of SMN in the spinal cord necessary for efficacy, and measured the efficiency of motor neuron transduction in the spinal cord after intrathecal delivery in pigs and nonhuman primates (NHPs). CNS injections of 5e10, 1e10, and 1e9 genome copies (gc) of self-complementary AAV9 (scAAV9)-hSMN1 into SMA mice extended their survival from 17 to 153, 70, and 18 days, respectively. Spinal cords treated with 5e10, 1e10, and 1e9 gc showed that 70-170%, 30-100%, and 10-20% of wild-type levels of SMN were attained, respectively. Furthermore, detectable SMN expression in a minimum of 30% motor neurons correlated with efficacy. A comprehensive analysis showed that intrathecal delivery of 2.5e13 gc of scAAV9-GFP transduced 25-75% of the spinal cord motor neurons in NHPs. Thus, the extent of gene expression in motor neurons necessary to confer efficacy in SMA mice could be obtained in large-animal models, justifying the continual development of gene therapy for SMA.

Distribution of nanoparticles throughout the cerebral cortex of rodents and non-human primates: Implications for gene and drug therapy.

When nanoparticles/proteins are infused into the brain, they are often transported to distal sites in a manner that is dependent both on the characteristics of the infusate and the region targeted. We have previously shown that adeno-associated virus (AAV) is disseminated within the brain by perivascular flow and also by axonal transport. Perivascular distribution usually does not depend strongly on the nature of the infusate. Many proteins, neutral liposomes and AAV particles distribute equally well by this route when infused under pressure into various parenchymal locations. In contrast, axonal transport requires receptor-mediated uptake of AAV by neurons and engagement with specific transport mechanisms previously demonstrated for other neurotropic viruses. Cerebrospinal fluid (CSF) represents yet another way in which brain anatomy may be exploited to distribute nanoparticles broadly in the central nervous system. In this study, we assessed the distribution and perivascular transport of nanoparticles of different sizes delivered into the parenchyma of rodents and CSF in non-human primates.

Gene therapy for misfolding protein diseases of the central nervous system.

Protein aggregation as a result of misfolding is a common theme underlying neurodegenerative diseases. Accordingly, most recent studies aim to prevent protein misfolding and/or aggregation as a strategy to treat these pathologies. For instance, state-of-the-art approaches, such as silencing protein overexpression by means of RNA interference, are being tested with positive outcomes in preclinical models of animals overexpressing the corresponding protein. Therapies designed to treat central nervous system diseases should provide accurate delivery of the therapeutic agent and long-term or chronic expression by means of a nontoxic delivery vehicle. After several years of technical advances and optimization, gene therapy emerges as a promising approach able to fulfill those requirements. In this review we will summarize the latest improvements achieved in gene therapy for central nervous system diseases associated with protein misfolding (e.g., amyotrophic lateral sclerosis, Alzheimer's, Parkinson's, Huntington's, and prion diseases), as well as the most recent approaches in this field to treat these pathologies.

Strong cortical and spinal cord transduction after AAV7 and AAV9 delivery into the cerebrospinal fluid of nonhuman primates.

The present study builds on previous work showing that infusion of adeno-associated virus type 9 (AAV9) into the cisterna magna (CM) of nonhuman primates resulted in widespread transduction throughout cortex and spinal cord. Transduction efficiency was severely limited, however, by the presence of circulating anti-AAV antibodies. Accordingly, we compared AAV9 to a related serotype, AAV7, which has a high capsid homology. CM infusion of either AAV7 or AAV9 directed high level of cell transduction with similar patterns of distribution throughout brain cortex and along the spinal cord. Dorsal root ganglia and corticospinal tracts were also transduced. Both astrocytes and neurons were transduced. Interestingly, little transduction was observed in peripheral organs. Our results indicate that intrathecal delivery of either AAV7 or AAV9 directs a robust and widespread cellular transduction in the central nervous system and other peripheral neural structures.

Advances in thin tissue Golgi-Cox impregnation: fast, reliable methods for multi-assay analyses in rodent and non-human primate brain.

In 1873 Camillo Golgi discovered a staining technique that allowed for the visualization of whole neurons within the brain, initially termed 'the black reaction' and is now known as Golgi impregnation. Despite the capricious nature of this method, Golgi impregnation remains a widely used method for whole neuron visualization and analysis of dendritic arborization and spine quantification. We describe a series of reliable, modified 'Golgi-Cox' impregnation methods that complement some existing methods and have several advantages over traditional whole brain 'Golgi' impregnation. First, these methods utilize 60-100µm thick brain sections, which allows for fast, reliable impregnation of neurons in rats (7-14 days) and non-human primates (NHP) (30 days) while avoiding the pitfalls of other 'rapid Golgi' techniques traditionally employed with thin sections. Second, these methods employ several common tissue fixatives, resulting in high quality neuron impregnation in brain sections from acrolein, glutaraldehyde, and paraformaldehyde perfused rats, and in glutaraldehyde perfused NHP brain tissue. Third, because thin sections are obtained on a vibratome prior to processing, alternate sections of brain tissue can be used for additional analyses such as immunohistochemistry or electron microscopy. This later advantage allows for comparison of, for example, dendrite morphology in sections adjacent to pertinent histochemical markers or ultrastructural components. Finally, we describe a method for simultaneous light microscopic visualization of both tyrosine hydroxylase immunohistochemistry and Golgi impregnation in the same tissue section. Thus, the methods described here allow for fast, high quality Golgi impregnation and conserve experimental subjects by allowing multiple analyses within an individual animal.

Safety and tolerability of magnetic resonance imaging-guided convection-enhanced delivery of AAV2-hAADC with a novel delivery platform in nonhuman primate striatum.

Degeneration of nigrostriatal neurons in Parkinson's disease (PD) causes progressive loss of aromatic l-amino acid decarboxylase (AADC), the enzyme that converts levodopa (l-DOPA) into dopamine in the striatum. Because loss of this enzyme appears to be a major driver of progressive impairment of response to the mainstay drug, l-DOPA, one promising approach has been to use gene therapy to restore AADC activity in the human putamen and thereby restore normal l-DOPA response in patients with PD. An open-label phase I clinical trial of this approach in patients with PD provided encouraging signs of improvement in Unified Parkinson's Disease Rating Scale scores and reductions in antiparkinsonian medications. However, such improvement was modest compared with the results previously reported in parkinsonian rhesus macaques. The reason for this discrepancy may have been that the relatively small volume of vector infused in the clinical study restricted the distribution of AADC expression, such that only about 20% of the postcommissural putamen was covered, as revealed by l-[3-(18)F]-α-methyltyrosine-positron emission tomography. To achieve more quantitative distribution of vector, we have developed a visual guidance system for parenchymal infusion of AAV2. The purpose of the present study was to evaluate the combined magnetic resonance imaging-guided delivery system with AAV2-hAADC under conditions that approximate the intended clinical protocol. Our data indicate that this approach directed accurate cannula placement and effective vector distribution without inducing any untoward effects in nonhuman primates infused with a high dose of AAV2-hAADC.

Adeno-associated virus serotype 9 transduction in the central nervous system of nonhuman primates.

Widespread distribution of gene products at clinically relevant levels throughout the CNS has been challenging. Adeno-associated virus type 9 (AAV9) vector has been reported as a good candidate for intravascular gene delivery, but low levels of preexisting antibody titers against AAV in the blood abrogate cellular transduction within the CNS. In the present study we compared the effectiveness of vascular delivery and cerebrospinal fluid (CSF) delivery of AAV9 in transducing CNS tissue in nonhuman primates. Both delivery routes generated similar distribution patterns, although we observed a more robust level of transduction after CSF delivery. Consistent with previous reports administering AAV9, we found greater astrocytic than neuronal tropism via both routes, although we did find a greater magnitude of CNS transduction after CSF delivery compared with intravascular delivery. Last, we have demonstrated that delivery of AAV9 into the CSF does not shield against AAV antibodies. This has obvious implications when developing and/or implementing any clinical trial studies.

Acute and chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administrations elicit similar microglial activation in the substantia nigra of monkeys.

Increasing evidence suggests a pivotal role for neuroinflammation in the pathogenesis of Parkinson disease, but whether activated microglia participate in disease progression remains unclear. To clarify this issue, we determined the numbers of activated microglial cells in the substantia nigra pars compacta and ventral tegmental area of monkeys subacutely and chronically exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Monkeys in the subacute MPTP treatment group were killed 1 week after the last MPTP injection; chronically treated monkeys were killed either 6 or 35 months after the last MPTP injection. Subacute MPTP administration induced loss of dopaminergic neurons in the substantia nigra pars compacta and ventral tegmental area and microglial activation in the same areas. Chronic MPTP treatment resulted in greater dopaminergic neuron depletion in both treatment groups. Both groups of chronic MPTP-treated monkeys showed increased numbers of activated microglial cells in the substantia nigra pars compacta that were similar to those of the subacute MPTP treatment group. These results indicate that microglial activation seems to be induced mainly by the toxic effects of MPTP and that it does not further progress once the toxin administration has been terminated. This suggests that the progressive degeneration of nigral cells in Parkinson disease may not necessarily be associated with progressively increased microglial activation.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine exposure fails to produce delayed degeneration of substantia nigra neurons in monkeys.

We assessed the presence of degenerating neurons in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) of parkinsonian monkeys. For this purpose, we used two histological markers of cellular death, Fluoro Jade B (FJB) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL). Eight monkeys were subacutelly treated with four to six 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injections (1-1.5 mg/kg, cumulative dose) and sacrificed 1 week and 11 months after last MPTP injection. Eight additional monkeys were chronically exposed to MPTP (4.5-15.3 mg/kg, cumulative dose) and sacrificed 6-35 months after last MPTP dose. Three intact monkeys served as controls. The number of tyrosine hydroxylase (TH)- and TUNEL-positive cells was quantified in SNpc and VTA and colocalization of FJB-positive and TUNEL-positive cells with neuronal (TH, NeuN, MAP2) and glial markers (human ferritin, GFAP) assessed on doubly labelled tissue sections. Only MPTP monkeys with 1-week survival displayed few doubly FJB-TH-labelled cells. Both groups of subacute MPTP monkeys, but not chronic MPTP monkeys, showed a significant increased number of TUNEL-positive cells in SNpc. TUNEL-positive cells exhibited morphological features and histological markers indicative of glial cells, whereas TUNEL/NeuN or TUNEL/MAP-2 colocalization was not observed. Our results indicate that MPTP treatment produced a nonapoptotic cell death of dopaminergic cells and the activation of the apoptotic cascade in glial cells. More importantly, we failed to demonstrate the existence of a delayed neurodegenerative process in the dopaminergic neurons after concluding MPTP injection thus, casting doubt on the validity of the "progressive model" created by repeated MPTP administration to monkeys.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) does not elicit long-lasting increases in cyclooxygenase-2 expression in dopaminergic neurons of monkeys.

To elucidate the role of the prostaglandin synthase cyclooxygenase-2 (Cox-2) and the mechanisms of dopaminergic (DA) neurodegeneration, monkeys were injected subacutely or chronically (n = 5/group) with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Chronically treated animals developed parkinsonian signs and were killed 6 months after the last treatment; tyrosine hydroxylase-expressing neurons decreased in all substantia nigra (SN) cell groups in both treatment groups. In untreated controls (n = 3), there was low Cox-2 expression in ventral SN DA neurons and high expression in ventral tegmental area neurons. In subacutely treated monkeys, Cox-2 expression increased in surviving DA cells, particularly in the ventrolateral SN. In chronically treated monkeys, enhanced Cox-2 expression appeared only in surviving ventral tegmental area and ventral SN neurons. Thus increased Cox-2 did not persist in other SN neurons after discontinuing 1-methyl-4-phenyl-1,2,36-tetrahydropyridine. Some DA neurons in treated but not control monkeys expressed the active nuclear form of phospho-c-Jun, but not the active form of nuclear factor-kappaB. We conclude that Cox-2 expression does not confer vulnerability to neurodegeneration in DA neurons and that it is unlikely that a subacute insult to DA neurons can perpetuate degeneration through Cox-2 activation. Other mechanisms, probably through the Jun N-terminal kinase cascade, lead to DA cell death in this model.