Browsing by Subject "NeuroD1"

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    NeuroD1-Mediated Astrocyte-to-Neuron Reprogramming for Spinal Cord Injuries
    (2022-09) Sorensen, Maggie
    Spinal cord injuries impact over 17,000 Americans yearly, often resulting in drastic symptoms, including loss of movement, loss/alteration of sensation, and intense pain. In the process of injury, Central Nervous System (CNS) neurons, which have limited regenerative capabilities after trauma, maintain damage. As of now, clinical treatment of these injuries focuses on rehabilitation, such as physical therapy. Success has been found with the transplantation of human embryonic pluripotent stem cells, but the ethical boundaries present as a barrier that would likely prevent this treatment from becoming widely available. Previous studies have shown cell reprogramming to be a potential treatment for SCIs. Astrocyte-to-neuron conversion can be promoted by overexpression of certain neuronal transcription factors, including NeuroD1. Astrocytes are common CNS glial cells that are known to proliferate upon injury and are proximal in lineage to neurons, making them a good target for neuronal conversion. An adeno-associated virus (AAV9) containing inverted NeuroD1 and fluorescent protein mRuby2 was injected into the spinal cord either immediately after the contusion (T1) or one-week post-contusion (T2) when reactive astrocytes are thought to be at the highest density. The control, AAV9 without NeuroD1, followed the same timeline. Histological analysis was completed in order to visualize the cell localization within the spinal cord sections 6 weeks post-injury. GFAP was used to tag astrocytes and NeuN was used for neurons. mRuby2 fluorescence is indicative of successful viral entry. Successful reprogramming would be demonstrated by similar localization of mRuby2 and neuron markers. While this localization is not perfectly indicative of reprogramming, it would provide preliminary support to these methods being effective in vivo.
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    Proneural transcription factor NeuroD1-mediated direct neuronal reprogramming - an AAV approach
    (2019-12) Radha, Swathi
    Direct cellular reprogramming to drive lineage switching from one differentiated cell type to another can be exploited to develop cell-based therapies for neurodegenerative diseases. In vivo reprogramming provides an attractive therapeutic strategy to circumvent the hurdles of immune rejection and ethical constraints associated with transplant-based therapy. Supporting glial cells of the CNS can be reprogrammed to neurons by targeted viral delivery of transcription factors and small molecules. Previous studies have demonstrated the effectiveness of a single proneural transcription factor NeuroD1 to drive reprogramming in the reactive glial lesions of Alzheimer’s disease and stroke. However, the ability of NeuroD1 (ND1) to promote a similar benefit in models of Parkinson’s disease (PD) has yet to be demonstrated. The current study aims to test the hypothesis that NeuroD1 delivered via an Adeno-Associated Virus (AAV) can promote reprogramming in striatal astrocytes to neurons in an in vivo PD model. The FDA-approved, clinically employed AAV-9 gene delivery platform was used to transduce non-dividing cells with minimal off-target effects. A two-part AAV-9 viral system was designed to express ND1 in astrocytes, driven by the GFAP promoter. First, ND1-mediated direct neuronal reprogramming was tested using a simple and scalable in vitro culture system. Primary astrocytes in vitro transduced with the AAV9-ND1 dual virus system display characteristics of immature neuroglial precursor stage, suggesting successful reprogramming. Second, the potential of ND1 to drive reprogramming was assessed in vivo in mice. Intracranial and intravenously delivered AAV-9 dual virus system driven by the GFAP promoter targets astrocytes and surprisingly, mature resident neurons in vivo. Finally, ND1-mediated in vivo reprogramming was assessed in a well-established chemically-induced 6-hydroxydopamine (6-OHDA) Parkinson’s disease mouse model. The 6-OHDA injury model provides insight into the novel application of the AAV-9 dual virus system to target astrocyte-to-neuron reprogramming as well as to target resident neurons for potential neuronal repair in Parkinson’s disease.