Lineage reprogramming glia into neurons
1 PhD project offered in the IPP summer call 2017
Direct lineage conversion of brain-resident cells such as glia into induced neurons is gaining enormous interest as novel strategy for cell-based brain repair. We have previously demonstrated that mouse astroglia can be reprogrammed into functional excitatory or inhibitory neurons depending on the choice of reprogramming factors. More recently, we could show that these factors can also reprogram glial cells into induced neurons in vivo. Functional integration of new neurons critically depends on the ability to flexibly alter their gene expression program in response to neural activity. While we and other have begun to understand the changes in gene expression during the glia-to-neuron reprogramming process, nothing is known whether glia-derived neurons possess the ability of endogenous neurons to adapt their gene expression in an activity-dependent manner. Here we aim at unraveling the ability of glia-derived neurons to respond to alterations in neural network activity both in vitro and in vivo as a prerequisite to meaningful integration into pre-existing circuits.
PhD project proposal: Activity-dependent gene regulation in induced Neurons
In the first part of the project we will culture astroglia from the postnatal cortex and transduce these with retroviruses encoding for Neurog2 or Ascl1/Dlx2 for conversion into induced glutamatergic and GABAergic neurons (Heinrich et al., 2011). After different times of differentiation, neurons will be exposed to treatments that alter neural activity: high K+ (40mM), glutamate receptor agonists, or electrical field stimulation using frequencies known to induce long-term plasticity. Subsequently, induced neurons will be harvested for FACS-purification and RNA-sequencing. Using bioinformatics analyses in collaboration with Dr. Tiwari (IMB), we will identify genes expressed differentially upon stimulation. These gene expression changes will be compared to those of primary neurons undergoing the same stimulations. This will allow us to identify potential differences in the ability of induced and endogenous neurons to adapt their gene expression programs. We hypothesize that inability to fully unfold an activity-dependent gene regulation maybe due to failure to erase epigenetic modifications that were placed earlier during gliogenesis. Identifying such loci will be of great importance to characterize epigenetic barriers of lineage conversion into fully functional neurons.
In the second part of the project we will investigate whether neurons derived from lineage converted glia in vivo have the ability to respond to network activity. Toward this aim, we will induce glia-to-neuron conversion by forced expression of Sox/Ascl1 (Heinrich et al., 2014) or Neurog2/Bcl2 (Gascón et al., 2016) in the cerebral cortex of adult mice following a mild stab wound injury. Concomitant with the reprogramming factors we will express Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) selectively either in induced neurons or the surrounding neuronal network. After allowing induced neurons to mature in vivo, we will activate DREADDs by their cognate ligand (CNO). We will then prepare brain slices to record from induced neurons (Heinrich et al., 2014) or endogenous neurons and collect cytoplasm for library preparation and RNA-sequencing (patch-seq) to compare alterations of the gene expression program between induced and endogenous neurons. The degree to which induced neurons are able to unfold an activity-dependent gene expression program will reveal to which extent these cells share the capacity for adaptive changes. Loci that fail to be activated in induced neurons will be compared to those identified in the in vitro part of the project. In a follow-up project we will then subject these loci to extensive analyses of their epigenetic state following gliogenesis and subsequent glia-to-neuron lineage conversion.
Publications relevant to the Project
Gascón S, Mureno E, Masserdotti G, Ortega F, Russo GL, Petrik D, Deshpande A, Heinrich C, Karow M, Roberston SP, Schroeder T, Beckers J, Irmler M, Berndt C, Friedmann Angeli JP, Conrad M, Berninger B and Götz M (2016). Identification and successful negotiation of a metabolic checkpoint in direct neuronal reprogramming. Cell Stem Cell, 18, 396-409
Heinrich C, Bergami M, Gascón S, Lepier A, Viganò F, Dimou L, Sutor B, Berninger B* and Götz M* (2014) Sox2-mediated conversion of NG2 glia into induced neurons in the injured adult cerebral cortex. Stem Cell Reports, 3, 1000-1014. * Co-senior authors.
Heinrich C, Gascón S, Masserdotti G, Lepier A, Sánchez R, Simon-Ebert T, Schroeder T, Götz M and Berninger B (2011). Direct conversion of astroglia from the postnatal mouse cerebral cortex into subtype specific functional neurons. Nature Protocols, 6, 214-228.