Final Report Summary - DOPANEW (Dopaminergic Neurons for Cell Therapy in Parkinson's Disease)
Replacement of lost dopaminergic neurons by neural transplantation with embryonic nigral tissue is a most promising approach to the treatment of Parkinson's disease (PD). Preclinical trials have demonstrated that embryonic ventral mesencephalic tissue can survive, give rise to physiologically active neurons that are capable of releasing dopamine, and form connections with host neurons. Following transplantation of human embryonic neurons into the brain of PD patients some amelioration of their motor symptoms were demonstrated. However, a highly variable response of patients to transplantation has been observed and, in some cases, dyskinesia is associated to the transfer of cells into patients. Without a better understanding of this variability in outcome, neural transplantation is unlikely to develop into a widely used therapy.
The greatest limitation to extensive testing and application of neural transplantation is probably the limited availability of human embryonic dopaminergic neurons. Human nigral grafts are only made up of 5%-10% of neurons that are destined to become dopaminergic neurons, the rest of the cells being other neuronal and glial cell types. Furthermore, only a small fraction of the cells destined to become dopaminergic neurons (estimated at around 5%-10%) actually survive the grafting procedure. Despite the reported clinical benefit of neural grafting in PD patients, only about 400 patients have been transplanted worldwide, in part because of the shortage of embryonic donor tissue. Thus, while these studies provide the proof-of-principle that cell therapy in Parkinson's disease is possible, it is evident that alternative strategies and sources for neurons have to be exploited. This projects aims at exploiting new strategies to overcome these shortcomings.
In this project DopaNew (website: http://www.ibdml.univ-mrs.fr/dopanew) we exploited two alternative dopaminergic cell populations that hold potential for Parkinson's disease. First, we analyzed the molecular regulation of dopaminergic differentiation in the adult mammalian forebrain. Second, we developed new strategies and tools for the enrichment of dopaminergic neurons from heterogeneous mixtures thereby depleting cultures from teratoma forming cells.
Based on a method that allows the highly efficient electroporation of transgenes into neural stem cells in the postnatal ventricular wall, namely in stem cells for dopaminergic and GABAergic neurons of the olfactory bulb we labeled specifically lateral (GABAergic) or dorsal (dopaminergic) stem cells of the system with GFP and isolate their offspring at different time points after electroporation. Subsequently, RNA was isolated from the samples, amplified and subjected to microarray analysis for both, expression of mRNAs and microRNAs. In the following, differences in mRNA expression between the two targeted cell populations (dorsal/lateral) as well as between differentiation stages in time were investigated. This led to an exhaustive picture of gene expression in the dopaminergic and GABAergic lineages in the forebrain in space and time at a so-far unseen resolution that represents the fundament of the here-proposed project. The entire gene expression dataset has been submitted to a specialized website (GEO) and is now accessible to all users.
Next, we performed functional studies on selected factors identified in this screen. We found that the transcription factors Zic1 and Zic2 are efficient repressors of dopaminergic fate in forebrain neurons. Genetic inactivation of both factors leads to the appearance of super-numerous dopaminergic neurons in the forebrain. In line with these results we found that the ventral homeodomain protein Vax1 is implicated in dopaminergic fate repression, thereby leading to another type of forebrain interneurons. Finally, we demonstrated the implication of small regulatory RNAs (microRNAs) in the adult neurogenic process. First, we showed that miR-7a is a suppressor of dopaminergic fate, thereby allowing the generation of other neuron classes. Second, we showed that en entire family of these small regulatory, the miR)-200 family, is important for the terminal differentiation of adult generated neurons.
We established in vitro neuronal differentiation of dopaminergic neurons from iPS. We screened these cultures for markers that allow identifying and ultimately isolating defined subpopulations within these heterogeneous cell mixes. We focused on one particular surface factor, integrin associated protein IAP that was able to enrich heterogeneous iPS neuron cultures for midbrain floor plate markers such as FOXA2, LMX1A, LMX1B and EN1. In contrast sorted cells lack expression of pluripotency markers, and differentiate into mature dopaminergic neurons in vitro. Finally we used transplantation studies to show that sorted cells provide functional recovery in a rat model of PD.
From a translational perspective, this work generated molecular tools that might allow the targeted differentiation of dopaminergic neurons in the forebrain. This, in turn, represents a first step for the use of forebrain dopaminergic neurons in therapeutic paradigms based on cell transplantation or the mobilization of intrinsic neuronal stem cell pools. Moreover, our work clearly emphasizes the utility for IAP based cell sorting of heterogeneous cell populations in order to meet safety and quality requirements for future therapeutic cell products in the context of Parkinson’s disease.
The greatest limitation to extensive testing and application of neural transplantation is probably the limited availability of human embryonic dopaminergic neurons. Human nigral grafts are only made up of 5%-10% of neurons that are destined to become dopaminergic neurons, the rest of the cells being other neuronal and glial cell types. Furthermore, only a small fraction of the cells destined to become dopaminergic neurons (estimated at around 5%-10%) actually survive the grafting procedure. Despite the reported clinical benefit of neural grafting in PD patients, only about 400 patients have been transplanted worldwide, in part because of the shortage of embryonic donor tissue. Thus, while these studies provide the proof-of-principle that cell therapy in Parkinson's disease is possible, it is evident that alternative strategies and sources for neurons have to be exploited. This projects aims at exploiting new strategies to overcome these shortcomings.
In this project DopaNew (website: http://www.ibdml.univ-mrs.fr/dopanew) we exploited two alternative dopaminergic cell populations that hold potential for Parkinson's disease. First, we analyzed the molecular regulation of dopaminergic differentiation in the adult mammalian forebrain. Second, we developed new strategies and tools for the enrichment of dopaminergic neurons from heterogeneous mixtures thereby depleting cultures from teratoma forming cells.
Based on a method that allows the highly efficient electroporation of transgenes into neural stem cells in the postnatal ventricular wall, namely in stem cells for dopaminergic and GABAergic neurons of the olfactory bulb we labeled specifically lateral (GABAergic) or dorsal (dopaminergic) stem cells of the system with GFP and isolate their offspring at different time points after electroporation. Subsequently, RNA was isolated from the samples, amplified and subjected to microarray analysis for both, expression of mRNAs and microRNAs. In the following, differences in mRNA expression between the two targeted cell populations (dorsal/lateral) as well as between differentiation stages in time were investigated. This led to an exhaustive picture of gene expression in the dopaminergic and GABAergic lineages in the forebrain in space and time at a so-far unseen resolution that represents the fundament of the here-proposed project. The entire gene expression dataset has been submitted to a specialized website (GEO) and is now accessible to all users.
Next, we performed functional studies on selected factors identified in this screen. We found that the transcription factors Zic1 and Zic2 are efficient repressors of dopaminergic fate in forebrain neurons. Genetic inactivation of both factors leads to the appearance of super-numerous dopaminergic neurons in the forebrain. In line with these results we found that the ventral homeodomain protein Vax1 is implicated in dopaminergic fate repression, thereby leading to another type of forebrain interneurons. Finally, we demonstrated the implication of small regulatory RNAs (microRNAs) in the adult neurogenic process. First, we showed that miR-7a is a suppressor of dopaminergic fate, thereby allowing the generation of other neuron classes. Second, we showed that en entire family of these small regulatory, the miR)-200 family, is important for the terminal differentiation of adult generated neurons.
We established in vitro neuronal differentiation of dopaminergic neurons from iPS. We screened these cultures for markers that allow identifying and ultimately isolating defined subpopulations within these heterogeneous cell mixes. We focused on one particular surface factor, integrin associated protein IAP that was able to enrich heterogeneous iPS neuron cultures for midbrain floor plate markers such as FOXA2, LMX1A, LMX1B and EN1. In contrast sorted cells lack expression of pluripotency markers, and differentiate into mature dopaminergic neurons in vitro. Finally we used transplantation studies to show that sorted cells provide functional recovery in a rat model of PD.
From a translational perspective, this work generated molecular tools that might allow the targeted differentiation of dopaminergic neurons in the forebrain. This, in turn, represents a first step for the use of forebrain dopaminergic neurons in therapeutic paradigms based on cell transplantation or the mobilization of intrinsic neuronal stem cell pools. Moreover, our work clearly emphasizes the utility for IAP based cell sorting of heterogeneous cell populations in order to meet safety and quality requirements for future therapeutic cell products in the context of Parkinson’s disease.