Periodic Reporting for period 2 - IMAGINE (In vivo Imaging Genesis and Circuit Integration of Interneurons Engineered from Glia)
Reporting period: 2023-07-01 to 2024-12-31
Processes that lead to dysfunction or even death of neurons are causing irreversible and devastating damage to the brain, and thereby gravely affect the life of millions of patients, the people close to them, and the societies in which they live. One approach followed by researchers and clinicians aims at replacing dysfunctional or degenerated neurons. Classically, such cell-based therapy has been conceived as grafting new neurons in the brain area or close to where the original neurons had degenerated. Indeed, animal studies as well as ongoing clinical trials indicate that cell replacement can be a viable strategy towards restoring lost brain function.
However, we are exploring an alternative strategy that is based on the plasticity of cells to change their cell identity by rewiring their gene expression programmes. This process is referred to as lineage reprogramming. Recent years have provided evidence for the possibility of converting brain support cells referred to as glia into induced neurons in vivo by imposing the expression of key regulators of cell fate such as transcription factors. These play often a key role in determining neuronal fate during embryonic development but can be experimentally reused to induce an alternative fate that is in demand because of disease. The conceptual advantage of such glia-to-neuron conversion for brain repair is that cells generated in this way have the same genetic makeup as all other cells around, and hence do not cause an immunogenic response. Moreover, it is possible to envisage that ultimately this strategy could become largely non-invasive, albeit there are still many technical challenges to be overcome prior to that.
However, before such approach could become a clinical reality, important questions need to be addressed experimentally. The molecular processes by which glial cells give up their original identity and adopt a neuronal fate remains by and large enigmatic. We need to know how similar induced neurons can become to those they ought to replace. Moreover, it is unclear how neurons induced from glia may integrate into the pre-existing circuits of non-neurogenic brain regions such as the cerebral cortex which normally do not accommodate new neurons ever during an entire lifetime. Finally, can they participate in cortical information processing and even restore dysfunctional cortical circuits? In the project IMAGINE, we aim at elucidating these questions.
However, we are exploring an alternative strategy that is based on the plasticity of cells to change their cell identity by rewiring their gene expression programmes. This process is referred to as lineage reprogramming. Recent years have provided evidence for the possibility of converting brain support cells referred to as glia into induced neurons in vivo by imposing the expression of key regulators of cell fate such as transcription factors. These play often a key role in determining neuronal fate during embryonic development but can be experimentally reused to induce an alternative fate that is in demand because of disease. The conceptual advantage of such glia-to-neuron conversion for brain repair is that cells generated in this way have the same genetic makeup as all other cells around, and hence do not cause an immunogenic response. Moreover, it is possible to envisage that ultimately this strategy could become largely non-invasive, albeit there are still many technical challenges to be overcome prior to that.
However, before such approach could become a clinical reality, important questions need to be addressed experimentally. The molecular processes by which glial cells give up their original identity and adopt a neuronal fate remains by and large enigmatic. We need to know how similar induced neurons can become to those they ought to replace. Moreover, it is unclear how neurons induced from glia may integrate into the pre-existing circuits of non-neurogenic brain regions such as the cerebral cortex which normally do not accommodate new neurons ever during an entire lifetime. Finally, can they participate in cortical information processing and even restore dysfunctional cortical circuits? In the project IMAGINE, we aim at elucidating these questions.
When attempting neuronal reprogramming of glial cells of the early postnatal cortex by the proneural transcription factor achaete scute complex-like 1 (Ascl1), we observed to our surprise that Ascl1 induced proliferation of oligodendrocyte progenitor cells (OPCs) rather than their conversion into induced neurons (Galante et al, Front Neurosci 2022). However, when we employed a new combination of reprogramming factors consisting of a phospho-site-deficient variant of (Ascl1SA6) and Bcl2, this resulted in the conversion of astrocytes in the cerebral cortex of young mice into neurons that exhibit hallmarks of an important subclass of cortical interneurons: expression of the calcium binding protein parvalbumin and electrophysiological properties of fast-spiking interneurons (Marichal et al, Sci Adv 2024). This work has led to further studies aimed at the molecular, cellular and network changes that occur during conversion of astrocytes into parvalbumin-positive, fast-spiking induced neurons. We have undertaken single cell transcriptomics to uncover the molecular changes induced by expression of Ascl1SA6 versus wild type Ascl1 (each in combination with Bcl2). This work required successful isolation of astrocytes (and OPCs) undergoing reprogramming from the cerebral cortex of mice and subsequent single cell RNA sequencing. This was performed so far at 4 and 14 days following induction of reprogramming. The transcriptomics data show a clear difference between the use of wild type and mutant Ascl1 and provides molecular insights into why the latter is considerably more neurogenic than the former but also has a greater capacity for inducing the expression of parvalbumin. In parallel we have conducted two-photon live imaging of cells undergoing neuronal reprogramming providing direct evidence for the authenticity of the glia-to-neuron conversion process. Also, this data highlights the rapid acquisition of neuronal morphological hallmarks upon reprogramming. Finally, using in vivo calcium imaging we have been able to observe the emergence of calcium activity in induced neurons and their recruitment into functional visual circuits.
We have generated single cell transcriptome data of astrocytes undergoing in vivo conversion into interneuron-like cells. We are currently analyzing these data to learn about the molecular underpinnings that underlie the highly distinct reprogramming potency of wild type and mutant Ascl1. This will be key to further improve in vivo reprogramming strategies in the cortex and other brain regions. Furthermore, our two-photon live imaging experiments underscore the authenticity of transcription factor-induced astrocyte-to-neuron conversion in vivo. Even more importantly, we have direct evidence for the recruitment of induced neurons into cortical circuits processing sensory information. Over the course of the project IMAGINE, we wish to determine the functional contribution made by induced neurons to sensory information processing cortical circuits.