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Unraveling and overcoming hurdles in direct neuronal reprogramming
Unraveling and overcoming hurdles in direct neuronal reprogramming
Discovering new approaches to replace lost neurons following brain damage, as for traumatic injury, is one of the major goals in the field of regenerative medicine. Direct neuronal conversion of glial cells into neurons is emerging as a powerful strategy to achieve neuronal replacement. Despite large progress in the field, major limitations still exist before bringing this approach toward clinical translation. Major hurdles encompass epigenetic, metabolic and environmental barriers, which impede the newly generated neurons to properly integrate into the injured brain parenchyma, to substitute the lost neuronal networks and to fully replace the endogenous neuronal counterpart. The pathological process includes a cascade of fast-occurring events, such as metabolic impairment, reactive oxygen species and inflammatory molecules production, cell death, reactive gliosis and recruitment of inflammatory cells, which can have devastating consequences for the survival of the endogenous and reprogrammed neurons. Thus, a deeper understanding of the interplay between these mechanisms and how key players in the injury environment regulate processes of cell fate decision is needed. An important aspect fundamental to functional glia-to-neuron conversion in the injured brain is the viral vector used, especially in regard to the inflammatory reaction elicited in the tissue. Indeed we could observe that different viral vectors, routinely used in neuronal reprogramming studies, could induce diverse responses in the environment, independently from the transgene expressed. In particular, we noticed that retrovirus and lentivirus-mediated reprogramming elicited a strong inflammatory reaction, characterized by microglia and astrocyte reactivity, and massive immune cells infiltration, still persisting at the time when neurons start appearing. Conversely, adeno-associated virus (AAV)-mediated neuronal conversion had much a milder impact on the activation of the glial cells, with minimal immune cells recruitment. As using AAV greatly improved the rate of neuronal conversion, specification, integration and survival, compared to retroviral approaches, the environment plays a critical role in this successful reprogramming. A secondary mechanism also associated with inflammation is reactive oxygen species (ROS) production. Indeed, astrocytes transitioning into neurons face a burst of ROS, which lead to drastic cell death by ferroptosis if not properly counteracted. Consequently, buffering ROS with scavengers and pro-survival genes could greatly ameliorate the conversion efficiency in vitro as well as in vivo. As ROS production is mostly related to functional metabolic changes, we investigated this so far neglected aspect of direct neuronal reprogramming. I first demonstrated that a metabolic switch from glycolysis to oxidative phosphorylation is an essential requirement for a successful conversion to occur, as inhibiting the function of the electron transport chain did not improve the process despite the decrease in ROS, but actually entirely blocked the conversion of glia into neurons. As we were further interested in understanding the roles played by the metabolism in the reprogramming paradigm, we decided to characterize the mitochondria proteome of astrocytes and neurons, to identify differences in the mito-proteome between these cell types. We identified proteins enriched to each cell type, highlighting metabolic pathways relevant for their specific physiological functions. Interestingly, some of the specific mitochondrial proteins analyzed were correctly up-regulated or down-regulated during the transition from astrocytes to neurons, but at a relatively late stage in the reprogramming process. This finding further confirmed that a remodeling in mitochondrial proteins, and consequently metabolic pathways, occurs during the reprogramming process, even if partial and temporally delayed compared to the burst of ROS which converting neurons face. Early dCas9-mediated overexpression of anti-oxidant proteins in converting astrocytes, specific to the neuronal mitochondria proteome, could greatly improve the speed and efficiency of astrocyte-to- neuron conversion. Thus, understanding how to properly modify converting glial cells into neurons, not only from an epigenetic, genetic and morphological point of view, is necessary. In fact evaluating the impact on direct neuronal reprogramming of extrinsic factors, such as viral vectors and the environmental inflammatory reaction, as well as intrinsic constraints, such as mitochondria remodeling, ROS production and metabolic switch, could greatly improve the quality of reprogrammed neurons. The aim of my thesis is thus to unravel mechanisms involving inflammation, mitochondria and metabolic remodeling, which could increase our understanding of the glia-to-neuron conversion process, overall improving direct neuronal reprogramming.
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Russo, Gianluca Luigi
2019
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Russo, Gianluca Luigi (2019): Unraveling and overcoming hurdles in direct neuronal reprogramming. Dissertation, LMU München: Graduate School of Systemic Neurosciences (GSN)
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Abstract

Discovering new approaches to replace lost neurons following brain damage, as for traumatic injury, is one of the major goals in the field of regenerative medicine. Direct neuronal conversion of glial cells into neurons is emerging as a powerful strategy to achieve neuronal replacement. Despite large progress in the field, major limitations still exist before bringing this approach toward clinical translation. Major hurdles encompass epigenetic, metabolic and environmental barriers, which impede the newly generated neurons to properly integrate into the injured brain parenchyma, to substitute the lost neuronal networks and to fully replace the endogenous neuronal counterpart. The pathological process includes a cascade of fast-occurring events, such as metabolic impairment, reactive oxygen species and inflammatory molecules production, cell death, reactive gliosis and recruitment of inflammatory cells, which can have devastating consequences for the survival of the endogenous and reprogrammed neurons. Thus, a deeper understanding of the interplay between these mechanisms and how key players in the injury environment regulate processes of cell fate decision is needed. An important aspect fundamental to functional glia-to-neuron conversion in the injured brain is the viral vector used, especially in regard to the inflammatory reaction elicited in the tissue. Indeed we could observe that different viral vectors, routinely used in neuronal reprogramming studies, could induce diverse responses in the environment, independently from the transgene expressed. In particular, we noticed that retrovirus and lentivirus-mediated reprogramming elicited a strong inflammatory reaction, characterized by microglia and astrocyte reactivity, and massive immune cells infiltration, still persisting at the time when neurons start appearing. Conversely, adeno-associated virus (AAV)-mediated neuronal conversion had much a milder impact on the activation of the glial cells, with minimal immune cells recruitment. As using AAV greatly improved the rate of neuronal conversion, specification, integration and survival, compared to retroviral approaches, the environment plays a critical role in this successful reprogramming. A secondary mechanism also associated with inflammation is reactive oxygen species (ROS) production. Indeed, astrocytes transitioning into neurons face a burst of ROS, which lead to drastic cell death by ferroptosis if not properly counteracted. Consequently, buffering ROS with scavengers and pro-survival genes could greatly ameliorate the conversion efficiency in vitro as well as in vivo. As ROS production is mostly related to functional metabolic changes, we investigated this so far neglected aspect of direct neuronal reprogramming. I first demonstrated that a metabolic switch from glycolysis to oxidative phosphorylation is an essential requirement for a successful conversion to occur, as inhibiting the function of the electron transport chain did not improve the process despite the decrease in ROS, but actually entirely blocked the conversion of glia into neurons. As we were further interested in understanding the roles played by the metabolism in the reprogramming paradigm, we decided to characterize the mitochondria proteome of astrocytes and neurons, to identify differences in the mito-proteome between these cell types. We identified proteins enriched to each cell type, highlighting metabolic pathways relevant for their specific physiological functions. Interestingly, some of the specific mitochondrial proteins analyzed were correctly up-regulated or down-regulated during the transition from astrocytes to neurons, but at a relatively late stage in the reprogramming process. This finding further confirmed that a remodeling in mitochondrial proteins, and consequently metabolic pathways, occurs during the reprogramming process, even if partial and temporally delayed compared to the burst of ROS which converting neurons face. Early dCas9-mediated overexpression of anti-oxidant proteins in converting astrocytes, specific to the neuronal mitochondria proteome, could greatly improve the speed and efficiency of astrocyte-to- neuron conversion. Thus, understanding how to properly modify converting glial cells into neurons, not only from an epigenetic, genetic and morphological point of view, is necessary. In fact evaluating the impact on direct neuronal reprogramming of extrinsic factors, such as viral vectors and the environmental inflammatory reaction, as well as intrinsic constraints, such as mitochondria remodeling, ROS production and metabolic switch, could greatly improve the quality of reprogrammed neurons. The aim of my thesis is thus to unravel mechanisms involving inflammation, mitochondria and metabolic remodeling, which could increase our understanding of the glia-to-neuron conversion process, overall improving direct neuronal reprogramming.