A potential compensatory role for endogenous striatal tyrosine hydroxylase-positive neurons in a nonhuman primate model of Parkinson's disease.

The possibility of enhancing endogenous brain repair following neurological disorders, such as Parkinson's disease (PD), is of considerable recent interest. One such mechanism may exist in the striatum as an upregulated population of tyrosine hydroxylase (TH)-immunoreactive neurons that appear after 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) lesions in nonhuman primates as well as in humans with PD. An intriguing possibility is that these endogenous neurons reflect a compensatory mechanism to mitigate the loss of striatal DA due to progressive destruction of the nigrostriatal pathway. The possibility of enhancing the number and function of this population is attractive; however, it is crucial to gain further information about these cells in order to comprehend more fully their possible therapeutic potential. The current research was designed to investigate the fate of this endogenous population in African green monkeys rendered parkinsonian by MPTP lesions. Specifically, we assessed changes in the numbers of striatal neurons expressing TH at differing stages of the toxin-induced behavioral disability and discovered a close relationship with symptom severity and striatal DA neuron numbers. Increased numbers of striatal TH-positive neurons were associated with MPTP treatment that produced parkinsonian symptoms compared to numbers of these neurons in MPTP-treated asymptomatic animals and untreated controls. Expression of striatal DA neurons peaked at the manifestation of symptoms in mild/moderate animals and remained stable in animals that were severely parkinsonian. Furthermore, in severely debilitated animals that improved after fetal dopaminergic grafts, we discovered a return to control levels of the endogenous population. Taken together, our results further support the concept that this population of DA neurons responds to variations in striatal DA tone and may serve as a compensatory mechanism to restore striatal DA levels in the context of significant depletion. Artificially manipulating this endogenous population could prove beneficial for PD treatment, especially for individuals in early disease stages.

Human neural stem cells survive long term in the midbrain of dopamine-depleted monkeys after GDNF overexpression and project neurites toward an appropriate target.

Transplanted multipotent human fetal neural stem cells (hfNSCs) significantly improved the function of parkinsonian monkeys in a prior study primarily by neuroprotection, with only 3%-5% of cells expressing a dopamine (DA) phenotype. In this paper, we sought to determine whether further manipulation of the neural microenvironment by overexpression of a developmentally critical molecule, glial cell-derived neurotrophic factor (GDNF), in the host striatum could enhance DA differentiation of hfNSCs injected into the substantia nigra and elicit growth of their axons to the GDNF-expressing target. hfNSCs were transplanted into the midbrain of 10 green monkeys exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine. GDNF was delivered concomitantly to the striatum via an adeno-associated virus serotype 5 vector, and the fate of grafted cells was assessed after 11 months. Donor cells remained predominantly within the midbrain at the injection site and sprouted numerous neurofilament-immunoreactive fibers that appeared to course rostrally toward the striatum in parallel with tyrosine hydroxylase-immunoreactive fibers from the host substantia nigra but did not mature into DA neurons. This work suggests that hfNSCs can generate neurons that project long fibers in the adult primate brain. However, in the absence of region-specific signals and despite GDNF overexpression, hfNSCs did not differentiate into mature DA neurons in large numbers. It is encouraging, however, that the adult primate brain appeared to retain axonal guidance cues. We believe that transplantation of stem cells, specifically instructed ex vivo to yield DA neurons, could lead to reconstruction of some portion of the nigrostriatal pathway and prove beneficial for the parkinsonian condition.

Survival and integration of neurons derived from human embryonic stem cells in MPTP-lesioned primates.

A human embryonic stem cell (HESC) line, H1, was studied after differentiation to a dopaminergic phenotype in vitro in order to carry out in vivo studies in Parkinsonian monkeys. To identify morphological characteristics of transplanted donor cells, HESCs were transfected with a GFP lentiviral vector. Gene expression studies were performed at each step of a neural rosette-based dopaminergic differentiation protocol by RT-PCR. In vitro immunofluorescence revealed that >90% of the differentiated cells exhibited a neuronal phenotype by ß-III-tubulin immunocytochemistry, with 17% of the cells coexpressing tyrosine hydroxylase prior to implantation. Biochemical analyses demonstrated dopamine release in culture in response to potassium chloride-induced membrane depolarization, suggesting that the cells synthesized and released dopamine. These characterized, HESC-derived neurons were then implanted into the striatum and midbrain of MPTP (1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine)-exposed monkeys that were triple immunosuppressed. Here we demonstrate robust survival of transplanted HESC-derived neurons after 6 weeks, as well as morphological features consistent with polarization, organization, and extension of processes that integrated into the host striatum. Expression of the dopaminergic marker tyrosine hydroxylase was not maintained in HESC-derived neural grafts in either the striatum or substantia nigra, despite a neuronal morphology and expression of ß-III-tubulin. These results suggest that dopamine neuronal cells derived from neuroectoderm in vitro will not maintain the correct midbrain phenotype in vivo in nonhuman primates, contrasted with recent studies showing dopamine neuronal survival using an alternative floorplate method.

Comparison of fetal mesencephalic grafts, AAV-delivered GDNF, and both combined in an MPTP-induced nonhuman primate Parkinson's model.

We combined viral vector delivery of human glial-derived neurotrophic factor (GDNF) with the grafting of dopamine (DA) precursor cells from fetal ventral mesencephalon (VM) to determine whether these strategies would improve the anti-Parkinson's effects in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys, an animal model for Parkinson's disease (PD). Both strategies have been reported as individually beneficial in animal models of PD, leading to clinical studies. GDNF delivery has also been reported to augment VM tissue implants, but no combined studies have been done in monkeys. Monkeys were treated with MPTP and placed into four balanced treatment groups receiving only recombinant adeno-associated virus serotype 5 (rAAV5)/hu-GDNF, only fetal DA precursor cells, both together, or a buffered saline solution (control). The combination of fetal precursors with rAAV5/hu-GDNF showed significantly higher striatal DA concentrations compared with the other treatments, but did not lead to greater functional improvement in this study. For the first time under identical conditions in primates, we show that all three treatments lead to improvement compared with control animals.

Effects of neonatal neural progenitor cell implantation on adult neuroanatomy and cognition in the Ts65Dn model of Down syndrome.

As much of the aberrant neural development in Down syndrome (DS) occurs postnatally, an early opportunity exists to intervene and influence life-long cognitive development. Recent success using neural progenitor cells (NPC) in models of adult neurodegeneration indicate such therapy may be a viable option in diseases such as DS. Murine NPC (mNPC, C17.2 cell line) or saline were implanted bilaterally into the dorsal hippocampus of postnatal day 2 (PND 2) Ts65Dn pups to explore the feasibility of early postnatal treatment in this mouse model of DS. Disomic littermates provided karyotype controls for trisomic pups. Pups were monitored for developmental milestone achievement, and then underwent adult behavior testing at 14 weeks of age. We found that implanted mNPC survived into adulthood and migrated beyond the implant site in both karyotypes. The implantation of mNPC resulted in a significant increase in the density of dentate granule cells. However, mNPC implantation did not elicit cognitive changes in trisomic mice either neonatally or in adulthood. To the best of our knowledge, these results constitute the first assessment of mNPC as an early intervention on cognitive ability in a DS model.

Direct reprogramming of human fibroblasts into dopaminergic neuron-like cells.

Transplantation of exogenous dopaminergic neuron (DA neurons) is a promising approach for treating Parkinson's disease (PD). However, a major stumbling block has been the lack of a reliable source of donor DA neurons. Here we show that a combination of five transcriptional factors Mash1, Ngn2, Sox2, Nurr1, and Pitx3 can directly and effectively reprogram human fibroblasts into DA neuron-like cells. The reprogrammed cells stained positive for various markers for DA neurons. They also showed characteristic DA uptake and production properties. Moreover, they exhibited DA neuron-specific electrophysiological profiles. Finally, they provided symptomatic relief in a rat PD model. Therefore, our directly reprogrammed DA neuron-like cells are a promising source of cell-replacement therapy for PD.

Cellular repair in the parkinsonian nonhuman primate brain.

Parkinson disease (PD) is a neurodegenerative disorder that provides a useful model for testing cell replacement strategies to rejuvenate the affected dopaminergic neural systems, which have been destroyed by aging and the disease. We first showed that grafts of fetal dopaminergic neurons can reverse parkinsonian motor deficits induced by the toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), validating the feasibility of cellular repair in a primate nervous system. Subsequent clinical trials in Parkinson patients showed encouraging results, including long-term improvement of neurological signs and reduction of medications in some patients. However, many experienced little therapeutic benefit, and some recipients experienced dyskinesias, suggesting a lack of regulated control of the grafts. We have since attempted to improve cell replacements by placing grafts in their correct anatomical location in the substantia nigra and using strategies such as co-grafting fetal striatal tissue or growth factors into the physiologic striatal targets. Moreover, the use of fetal cells depends on a variable supply of donor material, making it difficult to standardize cell quality and quantity. Therefore, we have also explored possibilities of using human neural stem cells (hNSCs) to ameliorate parkinsonism in nonhuman primates with encouraging results. hNSCs implanted into the striatum showed a remarkable migratory ability and were found in the substantia nigra, where a small number appeared to differentiate into dopamine neurons. The majority became growth factor-producing glia that could provide beneficial effects on host dopamine neurons. Studies to determine the optimum stage of differentiation from embryonic stem cells and to derive useful cells from somatic cell sources are in progress.

Human neural stem cells migrate along the nigrostriatal pathway in a primate model of Parkinson's disease.

Although evidence of damage-directed neural stem cell (NSC) migration has been well-documented in the rodent, to our knowledge it has never been confirmed or quantified using human NSC (hNSC) in an adult non-human primate modeling a human neurodegenerative disease state. In this report, we attempt to provide that confirmation, potentially advancing basic stem cell concepts toward clinical relevance. hNSCs were implanted into the caudate nucleus (bilaterally) and substantia nigra (unilaterally) of 7, adult St. Kitts African green monkeys (Chlorocebus sabaeus) with previous exposure to systemic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that disrupts the dopaminergic nigrostriatal pathway. A detailed quantitative analysis of hNSC migration patterns at two time points (4 and 7 months) following transplantation was performed. Density contour mapping of hNSCs along the dorsal-ventral and medial-lateral axes of the brain suggested that >80% of hNSCs migrated from the point of implantation to and along the impaired nigrostriatal pathway. Although 2/3 of hNSCs were transplanted within the caudate, <1% of 3x10(6) total injected donor cells were identified at this site. The migrating hNSC did not appear to be pursuing a neuronal lineage. In the striatum and nigrostriatal pathway, but not in the substantia nigra, some hNSCs were found to have taken a glial lineage. The property of neural stem cells to align themselves along a neural pathway rendered dysfunctional by a given disease is potentially a valuable clinical tool.

Behavioral improvement in a primate Parkinson's model is associated with multiple homeostatic effects of human neural stem cells.

Stem cells have been widely assumed to be capable of replacing lost or damaged cells in a number of diseases, including Parkinson's disease (PD), in which neurons of the substantia nigra (SN) die and fail to provide the neurotransmitter, dopamine (DA), to the striatum. We report that undifferentiated human neural stem cells (hNSCs) implanted into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated Parkinsonian primates survived, migrated, and had a functional impact as assessed quantitatively by behavioral improvement in this DA-deficit model, in which Parkinsonian signs directly correlate to reduced DA levels. A small number of hNSC progeny differentiated into tyrosine hydroxylase (TH) and/or dopamine transporter (DAT) immunopositive cells, suggesting that the microenvironment within and around the lesioned adult host SN still permits development of a DA phenotype by responsive progenitor cells. A much larger number of hNSC-derived cells that did not express neuronal or DA markers was found arrayed along the persisting nigrostriatal path, juxtaposed with host cells. These hNSCs, which express DA-protective factors, were therefore well positioned to influence host TH+ cells and mediate other homeostatic adjustments, as reflected in a return to baseline endogenous neuronal number-to-size ratios, preservation of extant host nigrostriatal circuitry, and a normalizing effect on alpha-synuclein aggregation. We propose that multiple modes of reciprocal interaction between exogenous hNSCs and the pathological host milieu underlie the functional improvement observed in this model of PD.

Apoptotic natural cell death in developing primate dopamine midbrain neurons occurs during a restricted period in the second trimester of gestation.

Natural cell death (NCD) by apoptosis is a normal developmental event in most neuronal populations, and is a determinant of the eventual size of a population. We decided to examine the timing and extent of NCD of the midbrain dopamine system in a primate species, as dopamine deficiency or excess has been implicated in several disorders. Genetic or environmental differences may alter the extent of NCD and predispose individuals to neurological or psychiatric diseases. In developing rats, NCD in the midbrain dopamine system has been observed to start at the end of gestation and peak in the postnatal period. In fetal monkey brains, apoptosis in midbrain DA neurons was identified histologically by chromatin clumping in tyrosine hydroxylase-positive cells, and confirmed by TUNEL and active caspase-3 staining. A distinct peak of NCD occurred at about E80, midway through gestation in this species. We estimate that at least 50% of the population may be lost in this process. In other brains we determined biochemically that the onset of apoptosis coincides with the time of greatest rate of increase of striatal DA concentration. Thus, marked apoptotic NCD occurs in the primate midbrain dopamine system half-way through gestation, and appears to be associated with the rapid developmental increase in striatal dopamine innervation.

Aging-related changes in the nigrostriatal dopamine system and the response to MPTP in nonhuman primates: diminished compensatory mechanisms as a prelude to parkinsonism.

Aging is the most prominent risk factor for Parkinson's disease. Yet, consensus of how advancing age may predispose the dopamine (DA) system to parkinsonism is lacking. Three age ranges of female rhesus monkeys, 8-9, 15-17, and 21-31 years, received unilateral DA depletion with intracarotid 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Morphological and biochemical analyses of DA-depleted and intact hemispheres revealed three primary findings: (1) The intact striatum exhibited age-related declines in dopamine (DA) and homovanillic acid (HVA) that were present by middle age; (2) In the MPTP-treated striatum, the compensatory increase in DA activity was absent in old monkeys; and (3) Age-associated morphological changes included declines in the density of tyrosine hydroxylase (TH) positive fibers in striatum, decreased nigral soma size, and optical density of TH, but no significant loss of neurons. These findings suggest that aging produces changes in the nigrostriatal DA system that approach the threshold for expression of parkinsonian features, and that progressive impairment of plasticity may be central to the role of aging in development of parkinsonism.

Neural stem cells implanted into MPTP-treated monkeys increase the size of endogenous tyrosine hydroxylase-positive cells found in the striatum: a return to control measures.

Neural stem cells (NSC) have been shown to migrate towards damaged areas, produce trophic factors, and replace lost cells in ways that might be therapeutic for Parkinson's disease (PD). However, there is very little information on the effects of NSC on endogenous cell populations. In the current study, effects of implanted human NSC (hNSC) on endogenous tyrosine hydroxylase-positive cells (TH+ cells) after treatment with 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) were explored in nonhuman primates. After MPTP damage and in PD, the primate brain is characterized by decreased numbers of dopamine neurons in the substantia nigra (SN) and an increase in neurons expressing TH in the caudate nucleus. To determine how implanted NSC might affect these cell populations, 11 St. Kitts African green monkeys were treated with the selective dopaminergic neurotoxin, MPTP. Human NSC were implanted into the left and right caudate nucleus and the right SN of eight of the MPTP-treated monkeys. At either 4 or 7 months after NSC implants, the brains were removed and the size and number of TH+ cells in the target areas were assessed. The results were compared to data obtained from normal untreated control monkeys and to the three unimplanted MPTP-treated monkeys. The majority of hNSC were found bilaterally along the nigrostriatal pathway and in the substantia nigra, while relatively few were found in the caudate. In the presence of NSC, the number and size of caudate TH+ cells returned to non-MPTP-treated control levels. MPTP-induced and hNSC-induced changes in the putamen were less apparent. We conclude that after MPTP treatment in the primate, hNSC prevent the MPTP-induced upregulation of TH+ cells in the caudate and putamen, indicating that hNSC may be beneficial to maintaining a normal striatal environment.

Striatal trophic factor activity in aging monkeys with unilateral MPTP-induced parkinsonism.

Striatal trophic activity was assessed in female rhesus monkeys of advancing age rendered hemiparkinsonian by unilateral intracarotid administration of MPTP. Three age groups were analyzed: young adults (8-9.5 years) n=4, middle-aged adults (15-17 years) n=4, and aged adults (21-31 years) n=7. Fresh frozen tissue punches of caudate nucleus and putamen were collected 3 months after MPTP treatment and assayed for combined soluble striatal trophic activity, brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). This time point was chosen in an effort to assess a relatively stable phase of the dopamine (DA)-depleted state that may model the condition of Parkinson's disease (PD) patients at the time of therapeutic intervention. Analyses were conducted on striatal tissue both contralateral (aging effects) and ipsilateral to the DA-depleting lesion (lesion x aging effects). We found that combined striatal trophic activity in the contralateral hemisphere increased significantly with aging. Activity from both middle-aged and aged animals was significantly elevated as compared to young adults. Following DA depletion, young animals significantly increased combined striatal trophic activity, but middle-aged and aged animals did not exhibit further increases in activity over their elevated baselines. BDNF levels in the contralateral hemisphere were significantly reduced in aged animals as compared to young and middle-aged subjects. With DA depletion, BDNF levels declined in young and middle-aged animals but did not change from the decreased baseline level in old animals. GDNF levels were unchanged with aging and at 3 months after DA depletion. The results are consistent with several conclusions. First, by middle age combined striatal trophic activity is elevated, potentially reflecting a compensatory reaction to ongoing degenerative changes in substantia nigra DA neurons. Second, in response to DA depletion, young animals were capable of generating a significant increase in trophic activity that was sustained for at least 3 months. This capacity was either saturated or was not sustained in middle-aged and aged animals. Third, the aging-related chronic increase in combined striatal trophic activity was not attributable to BDNF or GDNF as these molecules either decreased or did not change with aging.

Embryonic mesencephalic grafts increase levodopa-induced forelimb hyperkinesia in parkinsonian rats.

Recent observations from clinical trials of neural grafting for Parkinson's disease (PD) have demonstrated that grafted dopamine neurons can worsen dyskinesias in some graft recipients. This deleterious side effect reveals a new challenge for neural transplantation, that of elucidating mechanisms underlying these postgraft dyskinesias. One problem facing this challenge is the availability of a cost-effective and reliable animal model in which to pursue initial investigations. In the current study, we investigated the interaction of an embryonic ventral mesencephalic (VM) dopamine (DA) neuron graft on levodopa (LD)-induced dyskinetic movements in unilaterally 6-hydroxydopamine-lesioned rats. Rats were administered LD (levodopa-carbidopa, 50:5 mg/kg) twice daily for 6 weeks after either a sham graft or VM DA graft. Although a single solid graft of embryonic DA neurons can prevent progression of some lesioned-induced behavioral abnormalities such as LD-induced rotation and dystonia, it significantly increases hyperkinetic movements of the contralateral forelimb. This differential effect of grafted neurons on abnormal behavioral profiles is reminiscent of that reported in grafted patients with PD. Data from this study illustrate important similarities between this model of parkinsonism and PD in human patients that make it suitable for initial preclinical investigations into possible mechanisms underlying postgraft aggravation of dyskinetic movements.

Embryonic ventral mesencephalic grafts to the substantia nigra of MPTP-treated monkeys: feasibility relevant to multiple-target grafting as a therapy for Parkinson's disease.

Transplantation of embryonic dopamine (DA) neurons is being studied as an experimental replacement therapy for the DA-deficiency characteristic of Parkinson's disease. Some studies suggest that one of the limitations of this approach is that intrastriatal placement of implants fails to consistently restore completely normal movement. One potential cause of this suboptimal therapeutic outcome is that changes in the neural activity of several structures in the basal ganglia circuitry resulting from striatal DA depletion is not adequately normalized by graft-derived DA replacement in striatum alone. In the present study, we assessed the feasibility of grafting embryonic DA neurons into the substantia nigra (SN) of adult parkinsonian monkeys as an approach to restoration of the DA modulation of striatal-nigral afferents that is lost after degeneration of SN neurons. Sixteen St. Kitts African green monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) received implants of embryonic monkey ventral mesencephalon (VM), or sham implants, aimed at the rostral SN. At 6 months after grafting, staining for tyrosine hydroxylase (TH) indicated that grafted DA neurons survived at this site, albeit often in reduced numbers compared with VM grafts to striatum. Grafted neurons extended neurites into the parenchyma of the SN, but there was no evidence of lengthy extension of graft-derived neurites rostrally along the trajectory of the mesostriatal fiber system. A region-specific, modest increase in DA levels and TH-positive fiber density in the ventral-medial putamen was detected, accompanied by modest but significant decreases in parkinsonian behaviors at 5-6 months after grafting. Our findings support the view that grafting embryonic tissue to the SN is a feasible procedure in nonhuman primates that provides a modest but detectable benefit of its own. These results encourage the further development of multiple-target grafting strategies as a means of restoring modulation of anatomically widespread basal ganglia structures relevant to treatment of Parkinson's disease.