Neural stem cells reduce hippocampal tau and reelin accumulation in aged Ts65Dn Down syndrome mice.

Tau accumulation, in the form of neurofibrillary tangles (NFT), is an early neuropathological characteristic of Alzheimer's disease (AD) and early onset AD frequently seen in Down syndrome (DS). We investigated the presence of tau accumulation in the brains of aging DS mice using the Ts65Dn mouse model. All aged mice appeared to have substantial clusters of extracellular granules that were positive for tau and reelin, but not for amyloid-ß or APP. These clusters were found primarily in CA1 of the hippocampus. In addition, the aged trisomic DS mice had a significantly greater accumulation of extracellular tau/reelin granular deposits compared to disomic littermates. These granules were similar to those described by others who also found extracellular proteinous granules in the brains of non-DS mice engineered to model aging and/or AD. When neural stem cells (NSC) were implanted unilaterally into the hippocampus of the Ts65Dn mice, the tau/reelin-positive granules were significantly reduced in both trisomic and disomic mice. Our findings indicate that changes in tau/reelin-positive granules could be used as an index for neuropathological assessment in aging DS and AD. Furthermore, changes in granule density could be used to test the efficacy of novel treatments, such as NSC implantation. Lastly, it is speculated that the unique abilities of NSC to migrate and express growth factors might be a contributing factor to reducing tau/reelin accumulation in aging DS and AD.

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.

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.

Oligodendrocyte-type 2 astrocyte-derived trophic factors increase survival of developing dopamine neurons through the inhibition of apoptotic cell death.

Survival of embryonic dopamine (DA) neurons is extremely low (5-20%) following transplantation. Strategies to increase this survival are critical to the future of transplantation for Parkinson's disease. We demonstrate here that a factor(s) released from striatal oligodendrocyte-type 2 astrocytes (SO2A) greatly improves the survival and phenotype expression of mesencephalic DA neurons in culture while simultaneously decreasing the presence of apoptotic nuclear profiles, as detected by the TUNEL method and bisbenzamide/tyrosine hydroxylase double labeling. This SO2A-derived trophic factor(s) has minimal effects on glia and no effect on nondopaminergic mesencephalic neurons. The developmental period during which this SO2A trophic effect occurs (E14-18) coincides with the period when mesencephalic grafts are undergoing the highest rates of apoptosis, i.e., immediately following implantation. Therefore, SO2A-derived trophic factor(s) offers great potential for the augmentation of grafted DA neuron survival.

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.

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.

Efferent synaptic connections of dopaminergic neurons grafted into the caudate nucleus of experimentally induced parkinsonian monkeys are different from those of control animals.

This study investigated the question of whether grafted dopamine cells in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys form synapses and, if they do, whether their postsynaptic targets were the same as those in control monkeys or in previous studies in rats. Electron-microscopic single immunostaining was performed for tyrosine hydroxylase on vibratome sections prepared from the head of the caudate nucleus of controls and MPTP-treated African green monkeys (Cercopithecus aethiops sabaeus) that received a graft. Furthermore, correlated light- and electron-microscopic double immunostaining was carried out for tyrosine hydroxylase and calbindin in the same brain area of MPTP-treated plus grafted animals. In control monkeys, the majority (97%) of dopamine boutons terminate on spines that were also synaptic targets of immunonegative boutons forming asymmetric synaptic contacts: synaptic triads. In MPTP-treated, grafted animals, the majority of transplanted dopamine cells terminate on dendritic shafts (67%) and somata (32%), and only a few (1.33%) form axospine synapses. The results of the double immunostaining experiments indicated that these newly formed axosomatic and axodendritic synapses are associated with calbindin-immunoreactive, medium-sized, spiny striatonigral projection neurons. These observations indicate that: (1) dopamine from transplanted embryonic tissue acts via synaptic contacts on host neurons; (2) the primary synaptic targets of transplanted dopamine cells are not spines but dendrites and somata of host neurons; (3) these target neurons are the same as in control animals; and (4) comparing these observations with results of control and grafted rats, there are major species differences between rats and monkeys in the dopamine innervation of both control and transplanted animals.

Co-grafted embryonic striatum increases the survival of grafted embryonic dopamine neurons.

To enhance the current therapeutic benefit of dopamine (DA) neuron grafts in Parkinson's disease, strategies must be developed that increase both DA neuron survival and fiber outgrowth into the denervated striatum. Previous work in our laboratory has demonstrated that dopaminergic neurons grow to greater size when co-grafted with striatal cell suspensions and display extensive tyrosine hydroxylase-positive (TH+) projections, but no conclusion could be reached concerning enhancement of survival of grafted DA neurons. The aim of the present study was to characterize further the potential trophic effects of striatal co-grafts on grafted mesencephalic DA neuron survival. Unilaterally lesioned male Fischer 344 rats were grafted with either a suspension of mesencephalic cells or with both mesencephalic and striatal cell suspensions. Co-grafts were either mixed together or placed separately into the striatum. Lesioned rats receiving no graft served as controls. Rotational behavior was assessed following amphetamine challenge at 2 weeks prior to grafting and at 4 and 8 weeks following grafting. Only rats receiving co-grafts of nigral and striatal suspensions separated by a distance of 1 mm showed significant behavioral recovery from baseline rotational asymmetry. Both mixed and separate striatal co-grafts were associated with a doubling of DA neuron survival compared with solo mesencephalic grafts. In the mixed co-graft experiment, DA neurite branching appeared enhanced and TH-rich patches were observed, whereas with co-grafts that were separated, TH+ innervation of the intervening host striatum was increased significantly. These results provide the first evidence suggesting that nigral-striatal co-grafts, particularly those placed separately and in proximity to each other, increase both DA neuron survival and neurite extension from the mesencephalic component of the grafts.

In vivo expression of therapeutic human genes for dopamine production in the caudates of MPTP-treated monkeys using an AAV vector.

An adeno-associated virus (AAV) vector, expressing genes for human tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC), demonstrated significantly increased production of dopamine in 293 (human embryonic kidney) cells. This bicistronic vector was used to transduce striatal cells of six asymptomatic but dopamine-depleted monkeys which had been treated with the neurotoxin MPTP. Striatal cells were immunoreactive for the vector-encoded TH after stereotactic injection for periods up to 134 days, with biochemical effects consistent with dopamine biosynthetic enzyme expression. A subsequent experiment was carried out in six more severely depleted and parkinsonian monkeys. Several TH/aadc-treated monkeys showed elevated levels of dopamine near injection tracts after 2.5 months. Two monkeys that received a beta-galactosidase expressing vector showed no change in striatal dopamine. Behavioral changes could not be statistically related to the vector treatment groups. Toxicity was limited to transient fever in several animals and severe hyperactivity in one animal in the first days after injection with no associated histological evidence of inflammation. This study shows the successful transfection of primate neurons over a period up to 2.5 months with suggestive evidence of biochemical phenotypic effects and without significant toxicity. While supporting the idea of an in vivo gene therapy for Parkinson's disease, more consistent and longer lasting biochemical and behavioral effects will be necessary to establish the feasibility of this appraoch in a primate model of parkinsonism.

Pattern of synaptophysin immunoreactivity within mesencephalic grafts following transplantation in a parkinsonian primate model.

The majority of investigations into the degree of restoration of neural circuitry following transplantation of the embryonic ventral mesencephalon to the striatum have focused upon the particular neurochemical subtypes of the fibers exchanged between graft and host. Visualization of neurites of specific neurotransmitter type while informative regarding the specificity of graft-host interactions, vastly underrepresents overall synaptogenesis as it may occur in the grafting situation. The present approach of using a molecular marker characteristic of all normal, functional synapses provides broader information about the synaptic remodeling that occurs after tissue grafting. Synaptophysin (SY), an integral membrane protein of the synaptic vesicle, is a reliable marker of nerve terminal differentiation. Immunohistochemical staining with antibodies directed against SY and the dopamine synthetic enzyme tyrosine hydroxylase (TH) was used to assess overall synaptic differentiation as well as the relationship between SY immunoreactivity and the distribution of grafted dopamine (DA) neurons and processes in mesencephalic grafts and mesencephalic-striatal co-grafts implanted in the striatum of MPTP-treated African green monkeys. Grafted embryonic cerebellar tissue was used as a comparison graft type that does not normally exchange prominent direct projections with striatum. Dense pericellular arrays of SY-positive terminals were associated with TH-positive neurons in mesencephalic grafts. In mixed mesencephalic-striatal co-grafts, TH-positive fiber patches within the striatal portion of the graft demonstrated a high degree of correspondence with SY immunoreactivity. In contrast, grafts of cerebellar tissue did not display the same pattern of prominent pericellular arrays of SY staining. These observations suggest that functional synapses are abundantly present within grafted mesencephalon, and that these contacts are enriched in areas of the graft occupied by DA neurons. Implantation of an inappropriate striatal target, the cerebellum, results in visibly diminished innervation. The pattern of SY labeling observed suggests that tissue grafts are extensively innervated, probably both from extrinsic and intrinsic sources, and that the pattern and density of this innervation corresponds to the appropriateness of the graft-host interaction.

Upregulation of striatal D2 receptors in the MPTP-treated vervet monkey is reversed by grafts of fetal ventral mesencephalon: an autoradiographic study.

Although neural transplantation holds promise as a treatment for Parkinson's disease, parkinsonian primates have generally exhibited inconsistent and incomplete recovery of motor functions following intrastriatal grafting of fetal ventral mesencephalon. One possible contributing factor to this variable response is lack of appropriate integration of donor neurons with host striatal circuitry with the result that there is insufficient dopamine release and postsynaptic dopamine receptor activation. This issue was examined by measuring the effect of transplanting fetal ventral mesencephalon to the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated (MPTP) monkeys on striatal D2 receptor binding. One year after receiving MPTP, D2 receptor binding was upregulated in the dorsal and ventral striatum of African green monkeys. Grafting of fetal ventral mesencephalon to the dorsal striatum of MPTP-treated monkeys 9 months before sacrifice, eliminated the D2 receptor upregulation in dorsal, but not ventral, region. Dopamine concentration in dorsal striatum of grafted MPTP-treated monkeys was significantly higher than in that region of MPTP-treated non-grafted monkeys. In addition, dopamine concentration was significantly higher in dorsal compared to ventral striatum of grafted MPTP-treated monkeys. These data, in addition to those from a previous autoradiographic study on dopamine uptake site density in these monkeys, strongly supports the hypothesis that ectopically placed ventral mesencephalon not only produces, but maintains the release of sufficient levels of dopamine to restore postsynaptic dopamine transmission in regions influenced by graft-derived dopamine.

Intrastriatal grafts from multiple donors do not result in a proportional increase in survival of dopamine neurons in nonhuman primates.

We examined the potential for "double grafts," i.e., grafts from two donors in each recipient, to enhance the total number of ventral mesencephalic dopamine neurons that survive grafting in adult African green monkeys. Because dopamine cell survival in grafts represents a small percentage of the total number of neurons grafted, several human clinical trials recently have employed grafts of tissue from multiple donors (e.g., from two to eight embryos per host recipient) in attempts to increase the total number of dopamine neurons that survive in grafts. Presumably, this is intended to elevate dopamine levels by providing more dopamine neurons to the damaged brain to alleviate the symptoms of parkinsonism. While well-developed grafts with several thousand dopamine neurons were found in most recipient animals, we observed a reduced total number of tyrosine hydroxylase positive neurons in the grafts in spite of the presence of some double grafts that were larger than normal. The overall growth of the grafts was impressive; some grafts were so large that they spanned the full dorsoventral extent of the caudate nucleus, probably reflecting the fact that twice as much tissue was implanted in each drop site in comparison to our standard protocol. However, some animals revealed atypical patterns of neurite outgrowth that appeared limited to the grafted tissue, and at least one monkey revealed "amorphous" grafts generally lacking in cellular structure, which suggests a possible rejection phenomenon. These findings raise questions about the use of multiple donors and suggest that the likelihood of rejection and/or cell death may be enhanced, which is of potential importance in the design of grafting strategies for clinical applications.

Severe long-term 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism in the vervet monkey (Cercopithecus aethiops sabaeus).

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces parkinsonian neurochemical and functional deficits in human and non-human primates. The utility of MPTP-induced parkinsonism in monkeys as an animal model of Parkinson's disease would be greater if it produced a persistent and stable behavioural syndrome so that the effects of novel therapeutic treatments can be accurately assessed. Further, the claim that many species including primates spontaneously recover from MPTP is a significant difference from idiopathic Parkinson's disease. This experiment focused on the long-term (six months) persistence of behavioural deficits in severely and moderately parkinsonian monkeys. The severity of the syndrome was based on a quantitative and objective measure of parkinsonism. Adult male African green (vervet) monkeys (Cercopithecus aethiops sabaeus) were treated with MPTP (cumulative dose 2.5 mg/kg over five days), and six were saline-control treated. MPTP-treated subjects were examined in two groups: those that were severely parkinsonian ("severe" group, n = 11) and those that were moderately impaired ("moderate" group, n = 5) the month after treatment. Summary factor scores were examined reflecting abnormal ("parkinsonian") behaviour and normal "healthy" behaviour. Subjects that displayed severe parkinsonism the month after MPTP were found to show stable and severe parkinsonism for the time period studied. In contrast, the group of animals that initially were moderately parkinsonian did not show a stable deficit during the study. These data suggest that the initial severity of the deficit is an important predictor of outcome. None the less, stable parkinsonism can be observed in severely parkinsonian subjects despite variability in the severity of the impairment in response to MPTP treatment. Two moderately and three severely affected subjects were studied for more than six months and they appeared to show equivalent scores at six months compared with between 11 to 19 months after MPTP administration. MPTP-treatment in the vervet monkey can result in persistent long-term deficits and therefore provides an excellent phenomenological as well as neuropathological model of Parkinson's disease.

Metabolic energy capacity of dopaminergic grafts and the implanted striatum in parkinsonian nonhuman primates as visualized with cytochrome oxidase histochemistry.

Histochemistry for visualization of the mitochondrial enzyme cytochrome oxidase has been detect cellular and regional differences in brain metabolism. We have examined the pattern of cytochrome oxidase (CO) staining in grafts of embryonic ventral mesencephalic tissue, and in the implanted striatum, of MPTP-treated monkeys as one index of the functional activity of grafted tissue and its influence on the host brain. Four monkeys were selected for study based on interesting variations in dopamine (DA) neuron content of their bilateral grafts as demonstrated with tyrosine-hydroxylase (TH) immunocytochemistry. The results suggest that grafts rich in DA neurons increase the metabolic activity of the implanted striatum of DA-depleted monkeys, and that this improvement of local energy metabolism is greater in the vicinity of grafts containing greater numbers of DA neurons. In addition, the pattern of CO staining within tissue transplants indicates that DA neurons exhibit the highest rate of metabolic activity among all cell types contained in the ventral mesencephalic grafts, and that the transplants receive metabolically active innervation from outside or within the grafted tissue.

Identification of novel variants of trkC mRNA transcripts in brain of African green monkeys.

The distinct biological effects of neurotrophins are mediated in part through their binding to the high-affinity neurotrophin receptors represented by the Trk family of receptor tyrosine kinases. Using the technique of reverse transcriptase-polymerase chain reaction (RT-PCR), we cloned several partial cDNAs encoding trkA, trkB, and trkC from fetal brains of African green monkeys. Southern analysis of PCR products showed that the ventral tegmental area of adult monkey and ventral midbrains of fetal monkeys of E59, E81, E91, and E150 days of gestation expressed all three trk gene transcripts, whereas only trkB and trkC mRNAs were detectable in the adult substantia nigra. The nucleotide sequences of the cloned monkey trk cDNAs are highly homologous to their human counterparts, and we detected a splice variant of trkC that has recently been described in humans, but not in rodents. Moreover, sequencing of trkC cDNAs derived from four fetal monkey midbrains revealed two novel variants with single nucleotide substitution. A missense mutation (AAT to AGT) was identified in the codon corresponding to codon 361 of the deduced human TrkC sequence, converting an encoded Asn to Ser. The second variant involves a silent transition at the third nucleotide of the codon Gly 362 (GGC to GGA). Furthermore, three of the four potential alleles involving these two trkC variants were detected in these monkeys, indicating that a segregation of multiple trkC alleles occurs in a geographically contained population of feral monkeys.