Researchers at King’s College London have made progress in cell reprogramming by transforming glial cells into functional neurons directly in the brain. Using a modified variant of the transcription factor Ascl1 (Ascl1SA6) and the protein Bcl2, they increased the efficiency of this conversion and generated functional neurons with specific characteristics of FS/PV interneurons, which are important for the balance of brain circuits.
Our brain is made up of different types of cells that work together to ensure the proper functioning of the nervous system. These include neurons, which are responsible for transmitting information, and glial cells, which provide structural and functional support to neurons.
During life, some regions of the brain have little or no capacity to regenerate neurons, especially after damage or disease.
In light of this, scientists have been looking for innovative ways to “reprogram” glial cells, transforming them into new functional neurons to repair tissues and restore damaged brain circuits.
The idea of reprogramming glial cells into neurons had already been demonstrated in the laboratory (in vitro). However, direct application to the brains of living organisms (in vivo) poses significant challenges, including confirming that the new neurons originate from glial cells and not from other cell types in the environment.
To this end, researchers have been using advanced tools, such as genetic tracking, which allow them to track and confirm the origin of the reprogrammed cells.
One of the main focuses of the research is the use of the transcription factor Ascl1, a protein that controls the expression of genes involved in the development and specification of neurons.
Ascl1 has already been shown to be effective in transforming glial cells into neurons, but its efficiency and consistency depend greatly on the biological context and specific conditions.
Recently, scientists at King’s College London modified Ascl1 from mice to make it more efficient. This mutant variant, called Ascl1SA6, was created by altering six sites in the protein that would normally be regulated by phosphorylation, a chemical process that can limit its reprogramming ability.
With these alterations, the variant showed greater stability and effectiveness in inducing new neurons from astrocytes, a type of glial cell common in the brain.
In addition, the researchers combined Ascl1SA6 with another protein, Bcl2, known to aid cell survival. This combination not only increased the rate of conversion of glia into neurons but also generated highly functional cells capable of firing electrical signals and behaving like mature neurons.
The experiments revealed that most of the newly generated neurons (called induced neurons, or iNs) came from astrocytes, while a few originated from oligodendrocyte progenitor cells (OPCs).
Coexpression of Ascl1SA6 and Bcl2 efficiently reprograms postnatal cortical glia into iNs. (A) Experimental design. Retroviral constructs encoding Ascl1 or Ascl1SA6 and Bcl2 were injected into the cortex of wt (C57BL/6J) mice 5 days after birth. Immunohistochemical analysis was performed at 12 or 28 dpi. (B) Low-magnification image showing cells transduced by Ascl1 or Ascl1SA6/Bcl2 at 12 dpi. High-magnification images depicting Dcx expression in cotransduced cells. Note the acquisition of conspicuous neuronal morphology in Ascl1SA6/Bcl2 iNs (arrows). Cells transduced with Bcl2 alone (GFP+ only (green)) maintained glial morphology (arrowheads). (C) At 28 dpi, the vast majority of Ascl1SA6/Bcl2 iNs expressed NeuN, whereas Ascl1/Bcl2 iNs did not express this neuronal marker and maintained glial morphology. Open arrows indicate marker-negative cells. (D) The proportion of double-transduced cells expressing Dcx (general neuronal marker), NeuN (mature neuron marker), or both neuronal markers at 12 dpi (left graph) and double-transduced cells expressing NeuN (right graph) at 28 dpi. dpi = days after injection
In addition, these new neurons displayed specific characteristics of FS/PV interneurons (parvalbumin-associated fast inhibitory neurons), which play a crucial role in regulating brain activity and are frequently implicated in neuropsychiatric disorders such as schizophrenia and autism, as well as epilepsy.
The tests confirmed that the conversion of glia into neurons was authentic, that is, it did not involve pre-existing cells. This was confirmed using genetic markers and techniques that traced cell lineage.
In addition, it was observed that the new neurons exhibited functional electrical and chemical properties, demonstrating that they could integrate into the brain's circuitry.
Forced coexpression of Ascl1SA6 and Bcl2 converts postnatal cortical glia into GABA- and PV-expressing iNs. (A) Ascl1/Bcl2-transduced cells do not express GABA (left), in contrast to the acquisition of GABA expression in Ascl1SA6/Bcl2 iNs (right), 28 dpi. Arrows indicate GABA-depleted (inset empty arrow) or GABA-containing (inset filled arrow) iNs. (B) The proportion of doubly transduced cells expressing GABA after Ascl1/Bcl2 or Ascl1SA6/Bcl2 reprogramming at 28 dpi. (D) Analysis of PV expression in Ascl1/Bcl2-transduced (left) and Ascl1SA6/Bcl2 iNs at 28 dpi. Arrows indicate iNs lacking PV (inset, empty arrow) or containing PV (inset, filled arrow). (E) The proportion of double-transduced cells expressing PV at 28 dpi. (F) Ascl1/Bcl2-transduced cells (left) lack Pvalb mRNA, whereas Ascl1SA6/Bcl2 iNs (right) express Pvalb mRNA as early as 12 dpi. Empty arrows indicate marker-negative cells. (G) Quantification of Pvalb mRNA transcripts expressed as the total number of puncta detected per individual cell in Ascl1/Bcl2-transduced cells, Ascl1SA6/Bcl2-transduced cells, and surrounding endogenous PV interneurons.
Despite these advances, scientists noted that many of the reprogrammed neurons did not survive long-term, suggesting the need to improve the brain environment to support their maturation and integration.
Furthermore, the technique used in this study (retrovirus) can only target actively proliferating glial cells, limiting its application to developing or injured brains.
Future studies will need to explore how to reprogram fully mature astrocytes in intact brains, which may require additional interventions to overcome barriers imposed by cellular maturity.
This study represents an important step in transforming glial cells into functional neurons. The Ascl1SA6 variant is a promising tool for neuronal reprogramming, paving the way for new therapies aimed at restoring damaged brain circuits in conditions such as epilepsy, traumatic brain injury, and neurodegenerative diseases.
Although challenges remain, these findings highlight the potential of cellular engineering to revolutionize the treatment of neurological and psychiatric diseases in the future.
READ MORE:
Reprogramming astroglia into neurons with hallmarks of fast-spiking parvalbumin-positive interneurons by phospho–site–deficient Ascl1
Nicolás Marichal, Sophie Péron, Ana Beltrán Arranz, Chiara Galante, Franciele Franco Scarante, Rebecca Wiffen, Carol Schuurmans, Marisa Karow, Sergio Gascón, and Benedikt Berninger
SCIENCE ADVANCES. 25 Oct 2024. Vol 10, Issue 43
DOI: 10.1126/sciadv.adl5935
Abstract
Cellular reprogramming of mammalian glia to an induced neuronal fate holds the potential for restoring diseased brain circuits. While the proneural factor achaete-scute complex-like 1 (Ascl1) is widely used for neuronal reprogramming, in the early postnatal mouse cortex, Ascl1 fails to induce the glia-to-neuron conversion, instead promoting the proliferation of oligodendrocyte progenitor cells (OPC). Since Ascl1 activity is posttranslationally regulated, here, we investigated the consequences of mutating six serine phospho-acceptor sites to alanine (Ascl1SA6) on lineage reprogramming in vivo. Ascl1SA6 exhibited increased neurogenic activity in the glia of the early postnatal mouse cortex, an effect enhanced by coexpression of B cell lymphoma 2 (Bcl2). Genetic fate-mapping revealed that most induced neurons originated from astrocytes, while only a few derived from OPCs. Many Ascl1SA6/Bcl2-induced neurons expressed parvalbumin and were capable of high-frequency action potential firing. Our study demonstrates the authentic conversion of astroglia into neurons featuring subclass hallmarks of cortical interneurons, advancing our scope of engineering neuronal fates in the brain.
Comments