Angel Viñuela

Dopamine neurons implanted into people with Parkinson's disease survive without pathology for 14 years

Postmortem analysis of five subjects with Parkinsons disease 9–14 years after transplantation of fetal midbrain cell suspensions revealed surviving grafts that included dopamine and serotonin neurons without pathology. These findings are important for the understanding of the etiopathogenesis of midbrain dopamine neuron degeneration and future use of cell replacement therapies.

Long-Term Survival of Dopamine Neurons Derived from Parthenogenetic Primate Embryonic Stem Cells (Cyno-1) After Transplantation

Dopamine (DA) neurons can be derived from human and primate embryonic stem (ES) cells in vitro. An ES cell–based replacement therapy for patients with Parkinsons disease requires that in vitro–generated neurons maintain their phenotype in vivo. Other critical issues relate to their proliferative capacity and risk of tumor formation, and the capability of migration and integration in the adult mammalian brain. Neural induction was achieved by coculture of primate parthenogenetic ES cells (Cyno‐1) with stromal cells, followed by sequential exposure to midbrain patterning and differentiation factors to favor DA phenotypic specification. Differentiated ES cells were treated with mitomycin C and transplanted into adult immunosuppressed rodents and into a primate (allograft) with out immunosuppression. A small percentage of DA neurons survived in both rodent and primate hosts for the entire term of the study (4 and 7 months, respectively). Other neuronal and glial populations derived from Cyno‐1 ES cells showed, in vivo, phenotypic characteristics and growth and migration patterns similar to fetal primate transplants, and a majority of cells (>80%) expressed the forebrain transcription factor brain factor 1. No teratoma formation was observed. In this study, we demonstrate long‐term survival of DA neurons obtained in vitro from primate ES cells. Optimization of differentiation, cell selection, and cell transfer is required for functional studies of ES‐derived DA neurons for future therapeutic applications.

Inhibition of the Dopamine D1 Receptor Signaling by PSD-95 *,◆

Dopamine D1 receptors play an important role in movement, reward, and learning and are implicated in a number of neurological and psychiatric disorders. These receptors are concentrated in dendritic spines of neurons, including the spine head and the postsynaptic density. D1 within spines is thought to modulate the local channels and receptors to control the excitability and synaptic properties of spines. The molecular mechanisms mediating D1 trafficking, anchorage, and function in spines remain elusive. Here we show that the synaptic scaffolding protein PSD-95 thought to play a role in stabilizing glutamate receptors in the postsynaptic density, interacts with D1 and regulates its trafficking and function. Interestingly, the D1-PSD-95 interaction does not require the well characterized domains of PSD-95 but is mediated by the carboxyl-terminal tail of D1 and the NH2 terminus of PSD-95, a region that is recognized only recently to participate in protein-protein interaction. Co-expression of PSD-95 with D1 in mammalian cells inhibits the D1-mediated cAMP accumulation without altering the total expression level or the agonist binding properties of the receptor. The diminished D1 signaling is mediated by reduced D1 expression at the cell surface as a consequence of an enhanced constitutive, dynamindependent endocytosis. In addition, genetically engineered mice lacking PSD-95 show a heightened behavioral response to either a D1 agonist or the psychostimulant amphetamine. These studies demonstrate a role for a glutamatergic scaffold in dopamine receptor signaling and trafficking and identify a new potential target for the modulation of abnormal dopaminergic function.

Selection of Embryonic Stem Cell-Derived Enhanced Green Fluorescent Protein-Positive Dopamine Neurons Using the Tyrosine Hydroxylase Promoter Is Confounded by Reporter Gene Expression in Immature Cell Populations

Transplantation of mouse embryonic stem (mES) cells can restore function in Parkinson disease models, but can generate teratomas. Purification of dopamine neurons derived from embryonic stem cells by fluorescence‐activated cell sorting (FACS) could provide a functional cell population for transplantation while eliminating the risk of teratoma formation. Here we used the tyrosine hydroxylase (TH) promoter to drive enhanced green fluorescent protein (eGFP) expression in mES cells. First, we evaluated 2.5‐kilobase (kb) and 9‐kb TH promoter fragments and showed that clones generated using the 9‐kb fragment produced significantly more eGFP+/TH+ neurons. We selected the 9‐kb TH clone with the highest eGFP/TH overlap for further differentiation, FACS, and transplantation experiments. Grafts contained large numbers of eGFP+ dopamine neurons of an appropriate phenotype. However, there were also numerous eGFP+ cells that did not express TH and did not have a neuronal morphology. In addition, we found cells in the grafts representing all three germ layers. Based on these findings, we examined the expression of stem cell markers in our eGFP+ population. We found that a majority of eGFP+ cells were stage‐specific embryonic antigen‐positive (SSEA‐1+) and that the genetically engineered clones contained more SSEA‐1+ cells after differentiation than the original D3 mES cells. By negative selection of SSEA‐1, we could isolate a neuronal eGFP+ population of high purity. These results illustrate the complexity of using genetic selection to purify mES cell‐derived dopamine neurons and provide a comprehensive analysis of cell selection strategies based on tyrosine hydroxylase expression.

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 Parkinsons 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.

Fate Mapping and Lineage Analyses Demonstrate the Production of a Large Number of Striatal Neuroblasts After Transforming Growth Factor α and Noggin Striatal Infusions into the Dopamine‐Depleted Striatum

Infusion of transforming growth factor α (TGFα) into the adult dopamine (DA)‐depleted striatum generates a local population of nestin+/proliferating cell nuclear antigen (PCNA)+ newborn cells. The precise origin and fate of these new striatal cells are unknown, making it difficult to direct them for neural repair in Parkinsons disease. Experiments in rats using 5‐bromo‐2′‐deoxyuridine (BrdU) to label neural progenitor cells showed that during TGFα infusion in the DA‐depleted striatum, newborn striatal cells formed a homogeneous population of precursors, with the majority coexpressing nestin, Mash1, Olig2, and epidermal growth factor receptor, consistent with the phenotype of multipotent C cells. Upon TGFα pump withdrawal, the subventricular zone (SVZ) was repopulated by neuroblasts. Strikingly, during this period, numerous clusters of doublecortin+/polysialylated neuronal cell adhesion molecule+ neuroblasts were also produced in the ipsilateral medial striatum. In parallel, striatal BrdU+/glial fibrillary acidic protein+ astrocytes were generated, but no BrdU+/O4+/CNPase+ oligodendrocytes were generated. Infusion of the neuralizing bone morphogenetic protein antagonist noggin after TGFα pump withdrawal increased the neuroblast‐to‐astrocyte ratio among new striatal cells by blocking glial differentiation but did not alter striatal neurogenesis. At no time or treatment condition were differentiated neurons generated, including DA neurons. Using 6‐hydroxydopamine‐lesioned nestin‐CreERT2/R26R‐YFP mice that allow genetic fate‐mapping of SVZ nestin+ cells, we show that TGFα‐generated striatal cells originate from SVZ nestin+ precursors that confirmed data from the rats on the phenotype and fate of striatal nestin+/PCNA+ cells upon TGFα withdrawal. This work demonstrates that a large population of multipotent striatal C‐like cells can be generated in the DA‐depleted striatum that do not spontaneously differentiate into DA neurons.

Recent advances in cell-based therapy for Parkinson disease

In this review, the authors discuss recent advances in the field of cell therapy for Parkinson disease (PD). They compare and contrast recent clinical trials using fetal dopaminergic neurons. They attribute differences in cell preparation techniques, cell type specification, and immunosuppression as reasons for variable outcome and for some of the side effects observed in these clinical trials. To address ethical, practical, and technical issues related to the use of fetal cell sources, alternative sources of therapeutic dopaminergic neurons are being developed. The authors describe the progress in enrichment and purification strategies of stem cell-derived dopaminergic midbrain neurons. They conclude that recent advances in cell therapy for PD will create a viable long-term treatment option for synaptic repair for this debilitating disease.

Implanted reuptake-deficient or wild-type dopaminergic neurons improve ON l-dopa dyskinesias without OFF-dyskinesias in a rat model of Parkinson's disease

OFF-l-dopa dyskinesias have been a surprising side-effect of intrastriatal foetal ventral mesencephalic transplantation in patients with Parkinsons disease. It has been proposed that excessive and unregulated dopaminergic stimulation of host post-synaptic striatal neurons by the grafts could be responsible for these dyskinesias. To address this issue we transplanted foetal dopaminergic neurons from mice lacking the dopamine transporter (DATKO) or from wild-type mice, into a rat model of Parkinsons disease and l-dopa-induced dyskinesias. Both wild-type and DATKO grafts reinnervated the host striatum to a similar extent, but DATKO grafts produced a greater and more diffuse increase in extra-cellular striatal dopamine levels. Interestingly, grafts containing wild-type dopaminergic neurons improved parkinsonian signs to a similar extent as DATKO grafts, but provided a more complete reduction of l-dopa induced dyskinesias. Neither DATKO nor wild-type grafts induced OFF-l-dopa dyskinesias. Behavioural and receptor autoradiography analyses demonstrated that DATKO grafts induced a greater normalization of striatal dopaminergic receptor supersensitivity than wild-type grafts. Both graft types induced a similar downregulation and normalization of PEnk and fosb/Δfosb in striatal neurons. In summary, DATKO grafts causing high and diffuse extra-cellular dompamine levels do not per se alter graft-induced recovery or produce OFF-l-dopa dyskinesias. Wild-type dopaminergic neurons appear to be the most effective neuronal type to restore function and reduce l-dopa-induced dyskinesias.

Parkinsonian monkeys with prior levodopa-induced dyskinesias followed by fetal dopamine precursor grafts do not display graft-induced dyskinesias.

Clinical trials testing the hypothesis that fetal dopamine grafts would provide antiparkinsonian benefit in patients who had already developed side effects from their long‐term use of L‐dopa revealed, in some cases, the presence of dyskinesias even in the absence of L‐dopa. The form, intensity, and frequency of these dyskinesias were quite variable, but their manifestation slowed the clinical development of cell replacement therapies. Rodent models of graft‐induced dyskinesias (GIDs) have been proposed, but their accuracy in modeling GIDs has been questioned because they usually require amphetamine for their presentation. The present study attempted to model GIDs in parkinsonian monkeys and, for the first time, to test the effect of grafts on previously dyskinetic monkeys. Toward this end, monkeys were rendered parkinsonian with n‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and dyskinetic with levodopa. They then received intraputamenal grafts of fetal dopaminergic cells, control cerebellar cells, or vehicle bilaterally and were studied for 18 months. Dopaminergic cells were grafted in a manner designed to produce either “hot spot” or “widespread” striatal innervation. Although levodopa‐induced dyskinesias could be elicited postoperatively, GIDs were never observed in any animal at any time after grafting. Grafted monkeys were also challenged with levodopa but did not show any greater responses to these challenges than before grafting. These studies support the development of future dopamine neuron cell transplantation therapy‐based approaches, indicating that in relevant primate models with appropriate cell preparation methodology, with successful graft survival and putamenal dopamine innervation, there is no evidence of graft‐induced dyskinesias. J. Comp. Neurol. 525:498–512, 2017.