Author: Ziyuan Guo
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Published: 2015
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Neuronal loss accompanying reactive gliosis and scarring is the major cause of dysfunction after brain injury and in neurodegenerative disorders, which are difficult to reverse with existing treatment approaches. Besides loss of neurons, gliosis is a common pathological process after brain injury, involving the activation of glial cells to proliferate and become hypertrophic to occupy the injured brain areas. Glial scar inhibits brain functional recovery. The primary objective of this thesis is to demonstrate the proof-of-concept that converting reactive glial cells into functional neurons in injured or diseased brain may provide a potential new approach for brain repair. Glial cells, including astrocytes, NG2 cells, and microglia, undergo reactive response to injury in order to form a defense system against the invasion of micro-organisms and cytotoxins into surrounding tissue. However, once activated, many reactive glial cells will stay in the injury sites and secrete neuroinhibitory factors to prevent neuronal growth, eventually forming glial scar inside the brain. So far, there is little success in the attempt to reverse glial scar after its formation. My recent work demonstrates that reactive glial cells in the cortex of stab-injured or Alzheimer's disease (AD) model mice can be directly reprogrammed into functional neurons in vivo, through retroviral expression of a single neural transcription factor, NeuroD1 (Guo et al., 2014). Cortical slice recordings revealed both spontaneous and evoked synaptic responses in NeuroD1-converted-neurons, suggesting that they can integrate into local neural circuits. NeuroD1 expression was also able to reprogram cultured human cortical astrocytes into functional neurons. My studies therefore suggest that direct reprogramming of reactive glial cells into functional neurons in vivo could provide a possible approach for repair of injured or diseased brain.The majority of astrocyte-converted neurons are glutamatergic, raising a concern whether in vivo reprogramming might tilt the excitation-inhibition (E/I) balance. Therefore, we further demonstrate that co-expression of NeuroD1 with another neural transcriptional factor Dlx2 efficiently reprograms NG2 glial cells into GABAergic neurons both in vitro and in vivo. Interestingly, the NG2-converted GABAergic neurons in the striatum are different from those converted in the prefrontal cortex, suggesting a regional influence on in vivo reprogramming. Brain slice recordings show that the NG2-converted GABAergic neurons are fully functional, with some firing fast-spiking action potentials, a characteristic feature of parvalbumin interneurons. More importantly, we have regenerated both excitatory and inhibitory neurons in the same cortical region by reprogramming astrocytes into glutamatergic neurons and NG2 cells into GABAergic neurons. Thus, my studies demonstrate that different glial cells can be reprogrammed into distinct subtypes of neurons to keep E/I balance and help functional recovery. Although cellular reconstruction by direct reprogramming makes possible new avenues of treatment for neurodegenerative or neurological disorders, functional recovery may depend critically on specificity of neuronal projection. Due to limited neurons converted by retrovirus, we decided to employ adeno-associated virus (AAV), a powerful transgene system, to deliver transcriptional factors, which can reprogram glial cells into neurons in mouse brains more efficiently than retrovirus. More importantly, those newly generated neurons can extend their axons towards contralateral cortex through corpus callosum or towards ipsilateral thalamus to integrate into global neural network. The converted neurons can survive more than 8 months and maintain their connection. Another advantage of AAV deliver system is to be able to cross blood-brain-barrier without invasive surgeries. And reprogrammed neurons achieved by intravenous injection of AAV without surgeries will make our technique more convenient for future clinical applications.To conclude, my thesis proves in vivo glia-neuron conversion by proneural gene(s). Such reprogramming approach allows us to generate new neurons and simultaneously reduce reactive glial cells. Furthermore, we can rebalance excitation and inhibition through reprogramming different glial cells into distinct subtypes of neurons. At last, our newly converted neurons project their axons to precise sites where the endogenous neurons do. All these findings together indicate that our in vivo reprogramming might be a possible therapeutic approach for various neurological and neurodegenerative disorders.
