Optimizing the direct reprogramming of human glial cells into parvalbumin interneurons

Abstract

Inhibitory neurons or interneurons are important regulators of brain signaling. Parvalbumin (PV) interneurons, the most common interneuron subtype, have the unique ability to fire a rapid train of action potentials. They regulate the activity of pyramidal neurons to promote appropriate behavioral responses. Cortical PV interneurons’ function is greatly diminished in neuropsychiatric disorders e.g. Epilepsy, schizophrenia and autism. Therefore, there is a focus to develop new cell therapies to restore the function of PV interneurons, e.g. using stem cells.

Direct reprogramming is an emerging field of research that aims to reprogram non-neuronal cells into neurons by injecting specific neuronal genes. This is a new attractive approach to generate new human neurons. Our lab has demonstrated that human glial cells can be con-verted into neurons GABAergic interneurons and PV neurons. Glia cells are found through-out the brain and offer a promising target for in situ reprogramming. In our previous study, human glia was transduced with 5 transcription factors : Ascl1, Dlx5, Lhx6, Sox2, and Foxg1 (referred altogether to as ADLSF). However, more research is required to determine whether different reprogramming genes could improve the generation of PV interneurons and increase the yield of PV cells.

My project focused on testing novel transcription factors with the aim of improving the PV reprogramming. In addition to my project I compared two transcription regulation systems and investigated a new nanofiber scaffold that was hypothesized to provide a beneficial sup-port to the cell. Although the addition of the new factors showed a moderate effect on in-creasing neuronal markers and PV expression at the transcription level, high-content screen-ing quantifications remained unchanged. Additionally, the comparison of the two regulation systems revealed a slight improvement in PV expression with the inducible system. Fur-thermore, the study explored the compatibility of the nanofiber scaffold for glia reprogram-ming, demonstrating its effectiveness in generating PV interneurons. However, this project is subject to several limitations, notably a restricted number of replicates. These limitations are thoroughly discussed, and potential improvements and alternative methodologies are explored.

Popular Abstract

Neurons in the making!

Our brain is just like a well-organized team: different types of neurons have specific roles to ensure optimal function. Inhibitory interneurons supervise the activity of other excitatory neurons to maintain the electrical balance of the brain. Parvalbumin interneurons are key players in brain signaling. Damage or loss of these neurons is seen in multiple brain disorders, like schizophrenia or epilepsy. If we transform the brain’s own cells into parvalbumin neurons, we could regain the lost interneurons and treat brain disorders that affect millions worldwide.

To generate parvalbumin interneurons, we used the technique called direct reprogramming: with this emerging technique, we can rapidly change the fate of a cell. To do so, we force the cell to produce specific transcription factors, which regulate gene expression to achieve the wanted cell identity. Glial cells are supporting cells abundant in the brain and suitable for direct reprogramming. In the lab, we force them to express 5 different transcription factors. My thesis work has tested different strategies to improve the generation of parvalbumin interneurons from glial cells; we aimed for a stronger expression of parvalbumin and an increased number of parvalbumin interneurons. I did not work directly on brains (in vivo) but on glial cells grown in the lab (in vitro).

First, we tested two new transcription factors, used for the first time in reprogramming. They were added separately to our standard combination. As a result, the new combinations successfully transformed the glia cells into human neurons and tended to improve the parvalbumin neuron generation.

In a second experiment, we compared two different systems to regulate the 5 standard transcription factors: With one, the expression of all the factors can be switched on/off, with the other system, 3 factors are always expressed and 2 can be switched on/off. We wondered if this could affect the reprogramming. Surprisingly, both methods gave comparable results, although the inducible system hinted at an improvement in parvalbumin expression.

Then, we tested one final interesting approach. We used a miniature spiderweb-like scaffold to help the transformed cells grow better by mimicking the 3D structure of the brain. The cells were able to survive and could be reprogrammed efficiently in this structure. This scaffold holds potential to help the cells survive longer in culture since the interneurons are very fragile, and it could also improve cell maturation.

In conclusion, by manipulating the cell identity of glial cells, we could restore brain interneurons to cure diseases. To make this cell therapy possible, a long but exciting journey lies ahead!

Master’s Degree Project in Molecular Biology 45 credits 2024
Department of Biology, Lund University

Advisors: Daniella Rylander Ottosson, Christina-Anastasia Stamouli and Shayini Kidnapillai

• author: Vorgeat, Aurélie Delphine
• supervisor: Daniella Rylander; Christina-Anastasia Stamouli
• organization: Degree Projects in Molecular Biology
• course: MOBN02 20232
• year: 2024
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9176439
• date added to LUP: 2024-10-11 09:28:07
• date last changed: 2024-10-11 09:28:07