Susceptibility to a parkinsonian toxin varies during primate development.

Symptoms of Parkinson's disease typically emerge later in life when loss of nigrostriatal dopamine neuron function exceeds the threshold of compensatory mechanisms in the basal ganglia. Although nigrostriatal dopamine neurons are lost during aging, in Parkinson's disease other detrimental factors must play a role to produce greater than normal loss of these neurons. Early development has been hypothesized to be a potentially vulnerable period when environmental or genetic abnormalities may compromise central dopamine neurons. This study uses a specific parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to probe the relative vulnerability of nigrostriatal dopamine neurons at different stages of primate development. Measures of dopamine, homovanillic acid, 1-methyl-pyridinium concentrations and tyrosine hydroxylase immunoreactive neurons indicated that at mid-gestation dopamine neurons are relatively vulnerable to MPTP, whereas later in development or in the young primate these neurons are resistant to the neurotoxin. These studies highlight a potentially greater risk to the fetus of exposure during mid-gestation to environmental agents that cause oxidative stress. In addition, the data suggest that uncoupling protein-2 may be a target for retarding the progressive loss of nigrostriatal dopamine neurons that occurs in Parkinson's disease and aging.

Impact of methamphetamine on dopamine neurons in primates is dependent on age: implications for development of Parkinson's disease.

Methamphetamine is a CNS stimulant with limited therapeutic indications, but is widely abused. Short-term exposure to higher doses, or long-term exposure to lower doses, of methamphetamine induces lasting damage to nigrostriatal dopamine neurons in man and animals. Strong evidence indicates that the mechanism for this detrimental effect on dopamine neurons involves oxidative stress exerted by reactive oxygen species. This study investigates the relative susceptibility of dopamine neurons in mid-gestation, young, and adult (not aged) monkeys to four treatments with methamphetamine over 2 days. Primate dopamine neurons undergo natural cell death at mid-gestation, and we hypothesized that during this event they are particularly vulnerable to oxidative stress. The results indicated that at mid-gestation and in adults, dopamine neurons were susceptible to methamphetamine-induced damage, as indicated by loss of striatal tyrosine hydroxylase (TH) immunoreactivity and dopamine concentration. However, dopamine neurons in young animals appeared totally resistant to the treatment, despite this group having higher brain levels of methamphetamine 3 h after administration than the adults. As a possible explanation for the protection, striatal glial-derived neurotrophic factor (GDNF) levels were elevated in young animals 1 week after treatment, but not in adults following methamphetamine treatment. Implications of these primate studies are: (1) the susceptibility of dopamine neurons at mid-gestation to methamphetamine warns against the risk of exposing pregnant women to the drug or oxidative stressors, and supports the hypothesis of Parkinson's disease being associated with oxidative stress during development, (2) elucidation of the mechanism of resistance of dopamine neurons in the young animals to methamphetamine-induced oxidative stress may provide targets for slowing or preventing age- or disease-related loss of adult nigrostriatal dopamine (DA) neurons, and (3) the increased striatal production of GDNF in young animals, but not in adults, in response to methamphetamine, suggests the possibility of an age-related change in the neurotrophic capacity of the striatal dopamine system.

Aged monkeys as a partial model for Parkinson's disease.

Parkinson's Disease (PD) and the natural aging process share a number of biochemical mechanisms, including reduced function of dopaminergic systems. The present study aims to determine the extent that motor and behavioral changes in aged monkeys resemble parkinsonism induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. The behavioral and physiological changes in PD are believed to result largely from selective depletion of dopamine in the nigrostriatal system. In the present study, ten aged female monkeys were compared with three groups: 9 untreated young adult female monkeys, 10 young adult male monkeys and 13 older male monkeys that had been exposed to MPTP. Trained observers, blind as to age and drug condition and without knowledge of the hypotheses, scored the monkeys using the Parkinson's factor score (Parkscore), which has been validated by a high correlation with post mortem striatal dopamine (DA) concentrations. The aged animals had higher scores on the Parkscore compared with the young adults, with most of its component behavioral items showing significance (tremor, Eating Problems, Delayed initiation of movement, and Poverty of Movement). L-Dopa and DA-agonists did not clearly reverse the principal measure of parkinsonism. DA concentrations post mortem were 63% lower in 3 aged monkeys in the ventral putamen compared with 4 young adults, with greater reductions in putamen than in caudate (45%). We conclude that aged monkeys, unexposed to MPTP, show a similar profile of parkinsonism to that seen after the neurotoxin exposure to MPTP in young adult monkeys. The pattern of greater DA depletion in putamen than in caudate in aged monkeys is the same as in human Parkinson's disease and contrasts with the greater depletion in caudate seen after MPTP. Aged monkeys of this species reflect many facets of Parkinson's disease, but like older humans do not improve with standard dopamine replacement pharmacotherapies.

Efficient transduction of non-human primate motor neurons after intramuscular delivery of recombinant AAV serotype 6.

Retrograde transport of viral vectors in the rodent spinal cord provides a powerful means to administer a therapeutic transgene from the innervated musculature. With the aim of scaling up this approach to non-human primates, we have injected recombinant adeno-associated vectors (rAAV) serotype 6 expressing enhanced green fluorescent protein (eGFP) into the gastrocnemius muscle of African green monkeys to determine whether this results in efficient transgene delivery to lumbar motor neurons. Cells expressing eGFP were observed across more than 1 cm of the spinal cord 4 weeks after intramuscular injection, reaching more than half of motor neurons in some cross-sections. Furthermore, quantitative PCR on the spinal cord tissue confirmed that eGFP expression within motor neurons was due to bona fide retrograde transport of the vector genome from the muscle. Although infiltrations of macrophages and lymphocytes were observed in the rAAV2/6-injected muscle, there was no detectable immune response within the transduced region of the spinal cord. These findings imply that retrograde delivery of rAAV serotype 6 in a primate species constitutes a non-invasive and robust approach to transduce motor neurons, a crucial target cell population in neurodegenerative disorders, such as amyotrophic lateral sclerosis and spinal muscular atrophy.

Embryonic substantia nigra grafts in the mesencephalon send neurites to the host striatum in non-human primate after overexpression of GDNF.

In spite of partial success in treating Parkinson's disease by using ectopically placed grafts of dopamine-producing cells, restoration of the original neuroanatomical circuits, if possible, might work better. Previous evidence of normal anatomic projections from ventral mesencephalic (VM) grafts placed in the substantia nigra (SN) has been limited to neonatal rodents and double grafting or bridging procedures. This study attempted to determine whether injection of a potent growth-promoting factor, glial cell line-derived neurotrophic factor (GDNF), into the target regions or placement of fetal striatal co-grafts in the nigrostriatal pathway might elicit neuritic outgrowth to the caudate nucleus. Four adult St. Kitts green monkeys received embryonic VM grafts into the rostral mesencephalon near the host SN, and injections of adeno-associated virus 2 (AAV2)/GDNF or equine infectious anemia virus (EIAV)/GDNF into the caudate. Three adult monkeys were co-grafted with fetal VM tissue near the SN and fetal striatal grafts (STR) 2.5 mm rostral in the nigrostriatal pathway. Before sacrifice, the striatal target regions were injected with the retrograde tracer Fluoro-Gold (FG). FG label was found in tyrosine hydroxylase-labeled neurons in VM grafts in the SN of only those monkeys that received AAV2/GDNF vector injections into the ipsilateral striatum. All monkeys showed FG labeling in the host SN when FG labeling was injected on the same side. These data show that grafted dopaminergic neurons can extend neurites to a distant target releasing an elevated concentration of GDNF, and suggest that grafted neurons can be placed into appropriate loci for potential tract reconstruction.

Biocompatibility of PEG-based hydrogels in primate brain.

Degradable polymers have been used successfully in a wide variety of peripheral applications from tissue regeneration to drug delivery. These polymers induce little inflammatory response and appear to be well accepted by the host environment. Their use in the brain, for neural tissue reconstruction or drug delivery, also could be advantageous in treating neurodegenerative disorders. Because the brain has a unique immune response, a polymer that is compatible in the body may not be so in the brain. In the present study, polyethylene glycol (PEG)-based hydrogels were implanted into the striatum and cerebral cortex of nonhuman primates. Four months after implantation, brains were processed to evaluate the extent of astrogliosis and scaring, the presence of microglia/macrophages, and the extent of T-cell infiltration. Hydrogels with 20% w/v PEG implanted into the brain stimulated a slight increase in astrocytic and microglial/macrophage presence, as indicated by a small increase in glial fibrillary acidic protein (GFAP) and CD68 staining intensity. This increase was not substantially different from that found in the sham-implanted hemispheres of the brain. Staining for CD3+ T cells indicated no presence of peripheral T-cell infiltration. No gliotic scarring was seen in any implanted hemisphere. The combination of low density of GFAP-positive cells and CD68-positive cells, the absence of T cells, and the lack of gliotic scarring suggest that this level of immune response is not indicative of immunorejection and that the PEG-based hydrogel has potential to be used in the primate brain for local drug delivery or neural tissue regeneration.

Embryonic substantia nigra grafts show directional outgrowth to cografted striatal grafts and potential for pathway reconstruction in nonhuman primate.

Transplantation of embryonic dopamine (DA) neurons has been tested as a therapy for Parkinson's disease. Most studies placed DA neurons into the striatum instead of the substantia nigra (SN). Reconstruction of this DA pathway could serve to establish a more favorable environment for control of DA release by grafted neurons. To test this we used cografts of striatum to stimulate growth of DA axons from embryonic SN that was implanted adjacent to the host SN in African green monkeys. Embryonic striatum was implanted at one of three progressive distances rostral to the SN. Immunohistochemical analysis revealed DA neuron survival and neuritic outgrowth from the SN grafts at 12-36 weeks after grafting. Each animal showed survival of substantial numbers of DA neurons. Most fibers that exited SN grafts coursed rostrally. Striatal grafts showed evidence of target-directed outgrowth and contained dense patterns of DA axons that could be traced from their origin in the SN grafts. A polarity existed for DA neurites that exited the grafts; that is, those seen caudal to the grafts did not appear to be organized into a directional outflow while those on the rostral side were arranged in linear profiles coursing toward the striatal grafts. Some TH fibers that reached the striatal grafts appeared to arise from the residual DA neurons of the SN. These findings suggest that grafted DA neurons can extend neurites toward a desired target over several millimeters through the brain stem and caudal diencephalon of the monkey brain, which favors the prospect of circuit reconstruction from grafted neurons placed into appropriate locations in their neural circuitry. Further study will assess the degree to which this approach can be used to restore motor balance in the nonhuman primate following neural transplantation.

AAV2-mediated gene transfer of GDNF to the striatum of MPTP monkeys enhances the survival and outgrowth of co-implanted fetal dopamine neurons.

Neural transplantation offers the potential of treating Parkinson's disease by grafting fetal dopamine neurons to depleted regions of the brain. However, clinical studies of neural grafting in Parkinson's disease have produced only modest improvements. One of the main reasons for this is the low survival rate of transplanted neurons. The inadequate supply of critical neurotrophic factors in the adult brain is likely to be a major cause of early cell death and restricted outgrowth of fetal grafts placed into the mature striatum. Glial derived neurotrophic factor (GDNF) is a potent neurotrophic factor that is crucial to the survival, outgrowth and maintenance of dopamine neurons, and so is a candidate for protecting grafted fetal dopamine neurons in the adult brain. We found that implantation of adeno-associated virus type 2 encoding GDNF (AAV2-GDNF) in the normal monkey caudate nucleus induced overexpression of GDNF that persisted for at least 6 months after injection. In a 6-month within-animal controlled study, AAV2-GDNF enhanced the survival of fetal dopamine neurons by 4-fold, and increased the outgrowth of grafted fetal dopamine neurons by almost 3-fold in the caudate nucleus of MPTP-treated monkeys, compared with control grafts in the other caudate nucleus. Thus, the addition of GDNF gene therapy to neural transplantation may be a useful strategy to improve treatment for Parkinson's disease.

Influence of cell preparation and target location on the behavioral recovery after striatal transplantation of fetal dopaminergic neurons in a primate model of Parkinson's disease.

Surgeries involving transplantation of fetal dopamine (DA) neurons into the caudate-putamen of patients with Parkinson's disease (PD) have been performed in various clinical trials to examine a potential restoration of motor function. The absence of studies in non-human primates to define the best transplantation protocols have lead to the use of a broad variety of techniques that potentially could have a major impact on the clinical outcome. The effects of using different cell and tissue preparation, and surgical targets, remain unknown. For this purpose, 20 St. Kitts African Green Monkeys (AFG) rendered parkinsonian by i.m. injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were balanced into 4 groups and unilaterally grafted in the (a) caudate or (b) putamen with fetal ventral mesencephalic (VM) tissue as (c) solid pieces or as a (d) cell suspension. By 9 months post-transplantation all animals showed significant and similar behavioral improvement as determined by a UPDRS based PD scale. Postmortem analyses showed that VM transplants survived in all animals. They were located in both surgical target sites, producing a broad DA reinnervation of the targeted nuclei that could also extend to the non-grafted nucleus on the ipsilateral side. Although no differences between groups were found in survival of DA neurons or degree of DA reinnervation, there was a significant correlation between striatal reinnervation and behavioral recovery only in animals transplanted in the putamen surgical target. Additionally, there was in general a stronger glial reaction to solid grafts than to cell suspensions. These studies provide data for the optimal time course, cell preparation and surgical targets for systematic examinations of both potential benefits and side effects of dopamine neuron cell transplantation in primate models of PD.

AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in long-term TPP-I expression compatible with therapy for LINCL.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal, autosomal recessive disease resulting from mutations in the CLN2 gene with consequent deficiency in its product tripeptidyl peptidase I (TPP-I). In the central nervous system (CNS), the deficiency of TPP-I results in the accumulation of proteins in lysosomes leading to a loss of neurons causing progressive neurological decline, and death by ages 10-12 years. To establish the feasibility of treating the CNS manifestations of LINCL by gene transfer, an adeno-associated virus 2 (AAV2) vector encoding the human CLN2 cDNA (AAV2CUhCLN2) was assessed for its ability to establish therapeutic levels of TPP-I in the brain. In vitro studies demonstrated that AAV2CUhCLN2 expressed CLN2 and produced biologically active TPP-I protein of which a fraction was secreted as the pro-TPP-I precursor and was taken up by nontransduced cells (ie, cross-correction). Following AAV2-mediated CLN2 delivery to the rat striatum, enzymatically active TPP-I protein was detected. By immunohistochemistry TPP-I protein was detected in striatal neurons (encompassing nearly half of the target structure) for up to 18 months. At the longer time points following striatal administration, TPP-I-positive cell bodies were also observed in the substantia nigra, frontal cerebral cortex and thalamus of the injected hemisphere, and the frontal cerebral cortex of the noninjected hemisphere. These areas of the brain contain neurons that extend axons into the striatum, suggesting that CNS circuitry may aid the distribution of the gene product. To assess the feasibility of human CNS delivery, a total of 3.6 x 10(11) particle units of AAV2CUhCLN2 was administered to the CNS of African green monkeys in 12 distributed doses. Assessment at 5 and 13 weeks demonstrated widespread detection of TPP-I in neurons, but not glial cells, at all regions of injection. The distribution of TPP-I-positive cells was similar between the two time points at all injection sites. Together, these data support the development of direct CNS gene transfer using an AAV2 vector expressing the CLN2 cDNA for the CNS manifestations of LINCL.

Global gene and cell replacement strategies via stem cells.

The inherent biology of neural stem cells (NSCs) endows them with capabilities that not only circumvent many of the limitations of other gene transfer vehicles, but that enable a variety of novel therapeutic strategies heretofore regarded as beyond the purview of neural transplantation. Most neurodegenerative diseases are characterized not by discrete, focal abnormalities but rather by extensive, multifocal, or even global neuropathology. Such widely disseminated lesions have not conventionally been regarded as amenable to neural transplantation. However, the ability of NSCs to engraft diffusely and become integral members of structures throughout the host CNS, while also expressing therapeutic molecules, may permit these cells to address that challenge. Intriguingly, while NSCs can be readily engineered to express specified foreign genes, other intrinsic factors appear to emanate spontaneously from NSCs and, in the context of reciprocal donor-host signaling, seem to be capable of neuroprotective and/or neuroregenerative functions. Stem cells additionally have the appealing ability to 'home in' on pathology, even over great distances. Such observations help to advance the idea that NSCs - as a prototype for stem cells from other solid organs - might aid in reconstructing the molecular and cellular milieu of maldeveloped or damaged organs.

Quantitative analysis of transgene protein, mRNA, and vector DNA following injection of an adenoviral vector harboring glial cell line-derived neurotrophic factor into the primate caudate nucleus.

Gene therapy for neurodegenerative diseases relies on stable expression of a vector-mediated transgene in the human central nervous system (CNS). In nonhuman primate CNS, transgene expression has been primarily assessed using descriptive histological methods. Here, we quantified the expression of a human glial cell line-derived neurotrophic factor (hGDNF) transgene using an ELISA specific for hGDNF protein and real-time quantitative RT-PCR and PCR for hGDNF mRNA and vector DNA, respectively. Transgene expression was assessed 1 week after injection of an E1-, E3-deleted adenovirus harboring hGDNF into the caudate nucleus of St. Kitts green monkey. We found that 57-147 million and 116-771 million copies of hGDNF mRNA and vector DNA, respectively, were present per 10,000 copies of the beta-actin gene. In the same sites, 40-152 pg of hGDNF protein per milligram of tissue was measured. Comparisons of these measures among monkeys demonstrated variable vector DNA and protein levels, but consistent mRNA levels at one-third of the level of vector DNA. This suggests that local responses to the vector play a role in the level of transgene expression and that high levels of vector DNA do not necessarily predict a high level of transgene protein. However, the results of this study do show that neuroprotective levels of GDNF transgene expression can be achieved following injection of an adenoviral vector into nonhuman primate caudate. Moreover, these assays provide quantitative methods for evaluating and comparing viral vectors in primate CNS.

Estrogen is essential for maintaining nigrostriatal dopamine neurons in primates: implications for Parkinson's disease and memory.

There are sexual differences in several parameters of the nigrostriatal dopamine neurons, as well as in the progression of diseases associated with this system, e.g., Parkinson's disease and dementia. These differences, as well as direct experimental data in rodents, suggest that gonadal hormones play a role in modulating this system. To determine whether circulating estrogen might have long-term effects by altering the number of dopamine neurons, the density of dopamine neurons was calculated in the compact zone of the substantia nigra of male and intact female short- (10 d) and longer-term (30 d) ovariectomized and short- and longer-term ovariectomized but estrogen-replaced nonhuman primates (African green monkeys). Furthermore, the number of tyrosine hydroxylase-expressing neurons, the total number of all types of neurons, and the volume of the compact zone of the substantia nigra were calculated in 30 d ovariectomized and in 30 d ovariectomized and estrogen-replaced monkeys. Unbiased stereological analyses demonstrated that a 30 d estrogen deprivation results in an apparently permanent loss of >30% of the total number of substantia nigra dopamine cells. Furthermore, the density calculations showed that brief estrogen replacement restores the density of tyrosine hydroxylase-immunoreactive cells after a 10 d, but not after a 30 d, ovariectomy. Moreover, the density of dopamine cells is higher in females than in males. These observations show the essential role of estrogen in maintaining the integrity of the nigral dopamine system, suggest a new treatment strategy for patients with Parkinson's disease and with certain forms of memory-impairing disorders, and provide another rationale for estrogen replacement therapy for postmenopausal women.

Striatal dopaminergic correlates of stable parkinsonism and degree of recovery in old-world primates one year after MPTP treatment.

Despite widespread use of the primate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease, there is a paucity of data concerning the relationship between striatal dopaminergic function and behavior over time. This study examines the relationship between markers of dopamine neuron integrity and dopaminergic metabolic activity in striatal subregions with the degree of parkinsonian disability in 32 monkeys treated with MPTP one year earlier. Based on the parkinsonian summary score during the month following MPTP treatment, each monkey was assigned to one of four severity categories. We called these categories "Severe", "Moderate", "Mild" and "Asymptomatic". Monkeys in the Severe category were behaviorally stable, and loss of dopamine concentration was greater than 98% in all subregions of striatum one year after MPTP treatment. This value was not significantly different from the level of depletion, reported previously, at one to two months after MPTP in Severe monkeys, and apparently this loss of striatal dopamine is beyond the level from which effective compensations can occur. The parkinsonian disabilities in monkeys of other severity groups (Moderate, Mild, Asymptomatic) improved significantly over the year, despite having mean dopamine depletion of 75-99% in different subregions of striatum at one to two months after MPTP treatment. At one year after MPTP treatment, the mean dopamine depletions in different subregions of caudate nucleus and putamen had diminished in Asymptomatics (21-81%), Milds (35-96%), and Moderates (86-97%). Dopamine loss in nucleus accumbens was relatively spared compared with most striatal subregions, yet in Severe monkeys the decrease in this region reached 96%. In addition, at one year after MPTP treatment, there was a significant linear relationship between parkinsonian behavioral severity category and dopamine concentration, and homovanillic acid concentration and homovanillic acid/dopamine ratio in the striatum. The re-establishment of dopamine levels and homovanillic acid/dopamine ratios was most pronounced in putamen, ventromedial caudate nucleus and nucleus accumbens. Thus the small difference in striatal dopamine loss that distinguishes monkeys with widely different behavior at one to two months after MPTP increases over time. We suggest that the milder the initial loss, the greater capacity there is for regeneration or sprouting of dopamine terminals, which is reflected in marked increases in dopamine levels and modest elevations of metabolic activity (homovanillic acid/dopamine ratio). With greater initial losses, there is less capacity to increase terminal density, which is reflected later by smaller increases in striatal dopamine levels and more marked increases in metabolic activity. It appears that 5-10% of normal striatal dopamine levels is sufficient for overtly normal motor performance in non-human primates.

Inflammatory responses and their impact on beta-galactosidase transgene expression following adenovirus vector delivery to the primate caudate nucleus.

An E1, E3 deleted adenovirus vector, serotype 5, carrying the marker gene LacZ was bilaterally microinfused into the caudate nuclei of 10 St Kitts green monkeys. The location and number of cells expressing transgene and host immunologic response were evaluated at 1 week (n = 2) and 1 month (n = 8) following vector infusion. A large number of cells expressed beta-galactosidase in some monkeys, exceeding 600000 in one monkey, but no expression was seen in three of 10. All monkeys had positive adenoviral antibody titers before vector infusion, indicating the possibility of previous exposure to some adenovirus, but only one showed a significant increase in titer afterwards. Inflammatory cell markers revealed an inverse correlation between transgene expression and the extent of inflammatory response. Dexamethasone administered immediately before and for 8 days following vector delivery, however, had no effect on transgene expression. The demonstration of significant inflammatory responses in the brain of some individual primates, including demyelination, indicates the need for new generations of adenovirus vectors, or the successful suppression of inflammatory responses, before this vector is suitable for non-cytotoxic clinical applications in the CNS.

Spontaneous blink rates correlate with dopamine levels in the caudate nucleus of MPTP-treated monkeys.

Previous studies have suggested a dopaminergic regulation of eye blink rates in human and nonhuman primates. Blockade of either dopamine (DA) D1 or DA D2 receptors or DA depletion induced by the dopaminergic neurotoxin MPTP both decrease spontaneous eye blink rates in monkeys. MPTP-induced decreases in blink rates can be reversed by administration of the full efficacy D1 agonist dihydrexidine, which has also been found to have dramatic antiparkinsonian effects in MPTP-treated animals. Increases in blink rates can also be induced by D1 and D2 agonists in normal animals. In the current study, we have investigated whether blink rates correlate with concentrations of DA or HVA and/or HVA:DA ratios in specific brain regions in MPTP-treated monkeys. Furthermore, the potential relationship between the severity of behavioral indices of parkinsonism and blink rates were examined. We found that (1) blink rates significantly correlate positively with concentration of DA and inversely with HVA:DA ratios in the rostral portion of the ventromedial body of the caudate nucleus (CD), but not other subcortical regions, and (2) that severity of parkinsonism was inversely correlated with blink rate. These data support a dopaminergic regulation of blink rate and suggest that the ventromedial region of the body of the CD may be critically involved in regulation of blink rate.

Adenovirus-mediated transgene expression in nonhuman primate brain.

Transgene expression in the brain of St. Kitts green monkey, Cercopithecus aethiops sabeus, was studied following injection of a serotype 5 adenoviral vector deleted in E1 and E3. The vector harbored the transgene for Escherichia coli beta-galactosidase (beta-Gal) with the simian virus 40 (SV40) nuclear localization signal under control of the Rous sarcoma viral (RSV) long terminal repeat. Several titers ranging from 5 x 10(7) to 2 x 10(9) plaque-forming units (PFU) in volumes ranging from 5 to 250 microl were injected into the caudate nuclei of 18 monkeys. Monkeys were treated with dexamethasone for 9 days, beginning the day prior to surgery, and were sacrificed at 1 week or at 1, 2, or 3 months. At 1 week, beta-Gal was expressed in thousands of cells, including both neurons and astrocytes. In addition, some dopaminergic neurons in the substantia nigra expressed transgene, suggesting retrograde transport of the vector. At 1 month 162,000+/-68,000 (SEM) or 65,000+/-29,000 beta-Gal-expressing cells persisted in striatum injected with 6 x 10(8) PFU in 30 microl or 5 x 10(7) PFU in 5 microl, respectively. Transgene expression was also observed in one of two monkeys sacrificed at 2 months and in a single monkey sacrificed at 3 months. No transgene expression was observed at 1 month in striatum injected with a higher titer (2 x 10(9) PFU in 100 microl) or more dilute vector (5 x 10(7) PFU in 30 microl). Staining for the major histocompatibility complex II (MHC II) subtype DR showed intense staining in sites injected with a higher vector titer, in which no transgene persisted at 1 month, whereas low to moderate staining was present in sites with high transgene expression. These observations suggest that there is an optimal range of vector titers for obtaining persistent transgene expression from E1E3-deleted adenovirus in primate brain, above which host responses limit transgene stability.

Dopamine D4 receptor antagonist reversal of subchronic phencyclidine-induced object retrieval/detour deficits in monkeys.

D4 dopamine receptors (DRs) are enriched in the primate prefrontal cortex, a brain region implicated in cognitive processes, and mesoprefrontal dopaminergic systems appear to be involved in modulating some cognitive functions of the prefrontal cortex. Despite anatomical localization of D4 DRs within the frontal cortex, the role of these receptors, specifically, in the regulation of cognition or behavior in primates is unknown. In these studies, we sought to learn whether specific antagonism of D4 DRs would affect performance of a task dependent on the frontostriatal system. The effects of NGD94-1 (2-phenyl-4(5)-[4-(2-pyrimidinyl)-piperazin-1-yl)-methyl]-imidazol e dimaleate), a potent and selective D4 DR antagonist and haloperidol, a non-specific D2-like DR antagonist, on the performance of an object retrieval/detour task by monkeys were examined. The effects of these antagonists on the object retrieval task were evaluated in normal control monkeys and in subjects repeatedly exposed to phencyclidine (PCP), to induce frontal cortical dopaminergic and cognitive dysfunction. NGD94-1 (1-5 mg/kg) reversed the cognitive deficits of PCP pre-treated monkeys, whereas haloperidol (25 microg/kg) exacerbated PCP-induced performance impairments. A low dose of NGD94-1 failed to affect performance of control subjects, while both haloperidol and a high dose of NGD94-1 impaired control performance. These data show, for the first time, that D4 DRs modulate the cognitive functions of the frontostriatal system.