Neuronal conversion in the spinal cord dorsal horn

Open Access
- Author:
- Puls, Brendan William
- Graduate Program:
- Neuroscience
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 27, 2020
- Committee Members:
- Gong Chen, Dissertation Advisor/Co-Advisor
Kevin Douglas Alloway, Committee Member
Yingwei Mao, Dissertation Advisor/Co-Advisor
Gong Chen, Committee Chair/Co-Chair
Shaun Mahony, Outside Member
Kevin Douglas Alloway, Program Head/Chair
Yingwei Mao, Committee Chair/Co-Chair - Keywords:
- Spinal cord injury
NeuroD1
Astrocyte
Neuronal conversion
Mouse model
In vivo reprogramming - Abstract:
- Spinal cord injury (SCI) often leads to impaired motor and sensory functions, partially because the injury-induced neuronal loss cannot be easily replenished through endogenous mechanisms. In vivo neuronal reprogramming has emerged as a novel technology to regenerate neurons from endogenous glial cells by forced expression of neurogenic transcription factors. We have previously demonstrated successful astrocyte-to-neuron conversion in mouse brains with injury or a model of Alzheimer’s disease by overexpressing a single neurogenic transcription factor Neuronal differentiation 1 (NeuroD1) via retroviruses. In this dissertation, we demonstrate the regeneration of dorsal spinal cord neurons from reactive astrocytes after SCI via adeno-associated virus (AAV), a more clinically relevant gene delivery system. We find that NeuroD1 converts reactive astrocytes into neurons in the dorsal horn of stab-injured spinal cord with high efficiency (~95%). Interestingly, NeuroD1-converted neurons in the dorsal horn mostly acquire a glutamatergic neuronal subtype, expressing spinal neuron markers such as T-cell leukemia homeobox 3 (Tlx3) and Paired box 2 (Pax2) but not cortical neuron markers such as Forkhead box G1 (FoxG1) and T-box brain 1 (Tbr1), suggesting that the astrocytic lineage and local microenvironment affect the cell fate of conversion. Despite generating a glutamatergic majority, we show that the proportion of GABAergic neurons can be increased by the co-expression of an additional transcription factor Distal-less homeobox 2 (Dlx2) to control the excitatory/inhibitory balance of our converted neurons. Electrophysiological recordings show that the NeuroD1-converted neurons can functionally mature and integrate into local spinal cord circuitry by displaying repetitive action potentials and spontaneous synaptic responses. We further show that NeuroD1-mediated neuronal conversion can occur in the contusive SCI model, allowing future studies of evaluating this in vivo astrocyte-to-neuron conversion technology for the regeneration of gray matter and functional recovery after SCI.