Challenges and Efficacy of Astrocyte-to-Neuron Reprogramming in Spinal Cord Injury: In Vitro Insights and In Vivo Outcomes
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Abstract
Traumatic spinal cord injury (SCI) leads to the disruption of neural pathways, causing loss of neural cells, with subsequent reactive gliosis and tissue scarring that limit endogenous repair. One potential therapeutic strategy to address this is to target reactive scar-forming astrocytes with direct cellular reprogramming to convert them into neurons, by overexpression of neurogenic transcription factors. Here we used lentiviral constructs to overexpress Ascl1 or a combination of microRNAs (miRs) miR124, miR9/9*and NeuroD1 transfected into cultured and in vivo astrocytes. In vitro experiments revealed cortically-derived astrocytes display a higher efficiency (70%) of reprogramming to neurons than spinal cord-derived astrocytes. In a rat cervical SCI model, the same strategy induced only limited reprogramming of astrocytes. Delivery of reprogramming factors did not significantly affect patterns of breathing under baseline and hypoxic conditions, but significant differences in average diaphragm amplitude were seen in the reprogrammed groups during eupneic breathing, hypoxic, and hypercapnic challenges. These results show that while cellular reprogramming can be readily achieved in carefully controlled in vitro conditions, achieving a similar degree of successful reprogramming in vivo is challenging and may require additional steps.
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Summary
The manuscript comprises experimental approaches to induce transdifferentiation from astrocytes to neurons in a spinal cord injury model. The authors tested two lentivirus-based approaches targeting CNS developmental signalling pathways which were tested in both cortex- and spinal cord-derived cultured astrocytes.
Cortically-derived astrocytes were forced both to express neuronal markers such as NeuN, and to show electrophysiological responses consistent with mature neurons. Spinal cord-derived astrocytes proved to be less sensitive to the aforementioned transdifferentiation strategy.
Finally, they tested their lentiviral-based reprogramming in vivo and made electromyographic …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/14341006.
Summary
The manuscript comprises experimental approaches to induce transdifferentiation from astrocytes to neurons in a spinal cord injury model. The authors tested two lentivirus-based approaches targeting CNS developmental signalling pathways which were tested in both cortex- and spinal cord-derived cultured astrocytes.
Cortically-derived astrocytes were forced both to express neuronal markers such as NeuN, and to show electrophysiological responses consistent with mature neurons. Spinal cord-derived astrocytes proved to be less sensitive to the aforementioned transdifferentiation strategy.
Finally, they tested their lentiviral-based reprogramming in vivo and made electromyographic measurements of diaphragm responses in adult rats as a proxy of functional recovery after cervical SCI to investigate whether the lesion microenvironment would enhance the regenerative response.
Altogether, the results presented in the manuscript reveal a promising approach to astrocyte to neuron transdifferentiation. However, we consider several major concerns in the current version of the manuscript that need to be addressed before reaching its final form.
Major concerns
In the introduction the authors claim that: "Although limited, this in vivo reprogramming promoted a degree respiratory functional recovery of suggesting the therapeutic potential of this approach." We find it somewhat overstated as it is not clearly supported by the results. We suggest moderating the claim or thoroughly discussing it.
The introduction refers to an important methodological issue "... it has been challenging to differentiate between glial cells successfully reprogrammed into neurons and resident neurons that were inadvertently infected by viral vector injection." Nevertheless, the experimental approach does not address it. The manuscript will benefit from the discussion of this limitation to further clarify the relevance of the results.
We notice several figures are wrongly referenced in the main text. This needs to be addressed.
Spinal cord injury is at the core of the problem addressed in this manuscript. Furthermore, in vivo experiments were performed with spinal cord-derived astrocytes. We suggest the authors show the pharmacological analysis performed on spinal cord-derived astrocytes, as they show for cortically-derived astrocytes in Supplementary Figure 1.
Regarding the results provided in Figure 2, we find that the manuscript would benefit from clarifying the claim of GFAP expression in spinal cultures. In the current form of the images, it is not clearly visible for the Ascl1 group.
The electrophysiological analysis presented in Figures 1 and 2 would be enhanced if electrode order were considered. This would provide insight into network connectivity of the reprogrammed cells to the pre-existing network.
The experimental approach of cellular reprogramming using miRs+NeuroD1 is promising. However, the full significance of the results would be better understood if scrambled controls were included. We find it important to discuss this limitation in the manuscript.
In the methods section the authors declare they used "Adult, female, Sprague Dawley rats (n=42)" for in vivo experiments. The range of ages needs to be clarified. To better understand the context of the findings, we believe that the phase of the estrous cycle should be considered and clarified. The rationale behind the sample size is not obvious and needs to be explicit.
We find that some additional details regarding the RtTA control are needed. For instance, when the authors state "...control-treated cells that received rtTA (reverse tetracyclinecontrolled transactivator, C-rtTA,)" it is not clear what is the exact nature of this treatment.
Minor concerns
Figures 1I and 2F are somewhat confusing. We suggest reorganizing the layout to help the reader understand the results.
Additional details about the lentiviral vectors used in the study are advisable. We also suggest to include references to support the first claim of the results: "Given that Ascl1 (Addgene, #27150), and the combination of miR124, miR9/9* and NeuroD1 (miR124,miR9/9*+NeuroD1, Addgene, #31874, named as "miRs+ NeuroD1" from here on), have each demonstrated efficiency in converting different types of somatic cells into functional neurons; we initially tested their capacity to convert activated astrocytes into functional neurons in vitro".
Some legends do not comprehensively describe all elements of the figures. Some details are missing or insufficiently explained in their current form.
Reevaluate the inclusion of some elements of Supplementary Figure 1 as a main figure, considering the relevance of the data shown.
Competing interests
The authors declare that they have no competing interests.
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