Final Report Summary - REPROPARK (New experimental therapeutic approaches for Parkinson’s disease by direct DA neuronal reprogramming)
Limited access to human neurons has posed a significant barrier to progress in biological and preclinical studies of the human nervous system. In this prospective, neuronal reprogramming can offer the unique advantage to be fast and easy to replicate in large numbers in order to obtain iNeurons from multiple patients in a short time. Thus, these reprogramming technologies might have invaluable applications for the future modeling of idiopathic and sporadic disorders where many patients need to be sampled and their cells analyzed in comparative studies. In ReproPark, we established procedures for escalating the production of iNeurons and new protocols for generating novel neuronal and glial cell types. Through the assessment of multiple candidate transcription factor combinations, we defined minimal sets of TFs capable to reprogram mouse and human cells into iDA and iGABA neurons. These induced neurons acquired the relevant gene expression and morphological properties of the authentic counterparts. Similarly, we defined for the first time TF cocktails sufficient and necessary for the efficient reprogramming of adult skin cells into brain astrocytes and myelinating Schwann cells. These results provide strong evidence that supraphysiological expression of TFs controlling cell lineage differentiation during development promote efficient reprogramming of adult cells into distinct and specific neuronal and glial subtypes. By gene expression analysis, we identified Kdm2b as the main H3K4 trimethylase involved in direct neuronal cell conversion and its loss dramatically reduces the reprogramming process. In similar studies, we showed that L1 retrotransposable elements are re-expressed and mobilized during the initial phases of reprogramming to promote chromatin accessibility and gene transcription activation in target genes.
In ReproPark, we devised an innovative system to combine CRISPR/Cas9 gene targeting with direct neuronal reprogramming in order to specifically inactivate gene function only during the process of neuronal cell conversion. This represents a convenient and efficient single-step procedure to generate induced neurons with a targeted gene inactivation.
This program allowed us to explore new systems to culture human neurons to model brain circuits and synapse specificity. For this goal, we established long-term cultures of human neurons in a microfluidic platform to reconstitute the nigro-striatal pathway and the formation of functional DA synapses. The generation of functional in vitro circuitries with defined post- and pre-synaptic neuronal elements provides an accurate and sophisticated system to investigate both physiological and pathological processes. Further refinement of these technologies will lead to more elaborated designs to accommodate multiple neuronal populations for faithful and systematic modeling of complex brain networks.
Finally, ReproPark provided the first ever study describing the transplantation of iDA neurons in vivo, their functional integration in host brain networks and sustain a symptomatic rescue in PD mice. Thus, this work provides proof that direct neuronal reprogramming offers a method for obtaining stable and transplantable DA neurons suitable for cell replacement in a PD animal model. Moreover, we showed that DREADD technology can be efficiently applied to control activity of iNeurons in vivo. This combination is of extreme interest for DA cell replacement research and could pave the way for pharmacological approaches aimed at exerting efficient and noninvasive control of the grafted neuronal implant.
In ReproPark, we devised an innovative system to combine CRISPR/Cas9 gene targeting with direct neuronal reprogramming in order to specifically inactivate gene function only during the process of neuronal cell conversion. This represents a convenient and efficient single-step procedure to generate induced neurons with a targeted gene inactivation.
This program allowed us to explore new systems to culture human neurons to model brain circuits and synapse specificity. For this goal, we established long-term cultures of human neurons in a microfluidic platform to reconstitute the nigro-striatal pathway and the formation of functional DA synapses. The generation of functional in vitro circuitries with defined post- and pre-synaptic neuronal elements provides an accurate and sophisticated system to investigate both physiological and pathological processes. Further refinement of these technologies will lead to more elaborated designs to accommodate multiple neuronal populations for faithful and systematic modeling of complex brain networks.
Finally, ReproPark provided the first ever study describing the transplantation of iDA neurons in vivo, their functional integration in host brain networks and sustain a symptomatic rescue in PD mice. Thus, this work provides proof that direct neuronal reprogramming offers a method for obtaining stable and transplantable DA neurons suitable for cell replacement in a PD animal model. Moreover, we showed that DREADD technology can be efficiently applied to control activity of iNeurons in vivo. This combination is of extreme interest for DA cell replacement research and could pave the way for pharmacological approaches aimed at exerting efficient and noninvasive control of the grafted neuronal implant.