CHEMICAL REPROGRAMMING OF HUMAN ASTROCYTES TO MULTI-TYPES OF FUNCTIONAL NEURONS
Open Access
- Author:
- Yin, Jiuchao
- Graduate Program:
- Neuroscience
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 29, 2019
- Committee Members:
- Gong Chen, Dissertation Advisor/Co-Advisor
Gong Chen, Committee Chair/Co-Chair
Yingwei Mao, Committee Member
Richard W Ordway, Committee Member
Xiaojun Lance Lian, Outside Member - Keywords:
- astrocyte-to-neuron conversion
small molecule
chemical conversion
reprogramming
glutamatergic neuron
GABAergic neuron - Abstract:
- Neuronal loss accounts for the main symptoms in patients with neurodegenerative disorders and central nervous system (CNS) injuries. Functional recovery afterwards is hard to achieve because of limited adult neurogenesis in mammalian brains. Regenerating functional neurons, therefore, is essential for effective treatment of patients suffering from neuronal loss. Following nerve injury, astrocytes will proliferate and become reactive to form glial scar in order to protect neighboring tissues from further damage. The continuous presence of glial scar, however, also impedes functional recovery by inhibiting neuronal growth and axon regeneration in injured areas. Our lab has developed an innovative strategy to reprogram reactive astrocytes into functional neurons for CNS repair. In this dissertation, we report two different chemical formulas that can reprogram cultured human astrocytes into functionally distinct glutamatergic and GABAergic neurons. I have initially participated in our first project that established a chemical reprogramming method to convert human astrocytes directly into functional neurons with nine small molecules. However, 9-molecule cocktail will be difficult for translational applications. Here, we report a more efficient protocol with only four or even three chemicals to enable human astrocyte-to-neuron conversion. The chemically induced neurons (iNs) can survive more than 7 months in culture, fire repetitive action potentials, and display robust synaptic burst activities. Interestingly, cortical astrocyte-converted neurons are mostly glutamatergic, while midbrain astrocyte-converted neurons can yield some GABAergic neurons in addition to glutamatergic neurons. When administered in vivo through intracranial or intraperitoneal injection, the four-drug combination can significantly increase adult hippocampal neurogenesis. Mechanistically, we illustrate that transcriptional activation of neural transcription factors such as NEUROD1 and NGN2, together with an increase of MECP2 and a decrease of REST, may be critical for chemical reprogramming. In addition, through substituting each individual chemical with several different functional analogs, we demonstrate that inhibition of four signaling pathways TGFβ, BMP, GSK3β and Notch is crucial for changing an astrocyte into a neuron. Interestingly, the presence of N2 supplement in conversion medium, or to be more specific, insulin in N2 supplement is also critical to efficient astrocyte-to-neuron conversion. In an effort to induce inhibitory GABAergic neurons from cortical astrocytes, we tested signaling pathways related to GABAergic neuron specification during development. We demonstrate that modulation of SHH and FGF8 pathways, together with TGFβ, BMP, GSK3β and Notch pathways can reprogram human astrocytes to GABAergic neurons. Chemically converted GABAergic iNs progressively become functional neurons and form synaptic networks in culture. They develop forebrain deep layer neuron identity and hippocampal neuron identity, and co-express subtype-specific markers calbindin and calretinin. Importantly, during chemical conversion, we detect not only gradual expression of neuronal genes, but also sequential activation of transcription factors that are related to GABAergic neuron specification, including GSX2, ASCL1, DLX2, and GAD67, indicating the progressive identity acquisition toward GABAergic neurons. In summary, we identified two distinct small molecule protocols that can reprogram human astrocytes to excitatory glutamatergic neurons and inhibitory GABAergic neurons respectively. In spite of potential challenges in translating bench work to clinical applications, our work brings us one step further toward developing drug therapy for the treatment of neurological disorders.