Christophe Heinrich

Directing Astroglia from the Cerebral Cortex into Subtype Specific Functional Neurons

Forced expression of single defined transcription factors can selectively and stably convert cultured astroglia into synapse-forming excitatory and inhibitory neurons.

Reprogramming of Pericyte-Derived Cells of the Adult Human Brain into Induced Neuronal Cells

Reprogramming of somatic cells into neurons provides a new approach toward cell-based therapy of neurodegenerative diseases. A major challenge for the translation of neuronal reprogramming into therapy is whether the adult human brain contains cell populations amenable to direct somatic cell conversion. Here we show that cells from the adult human cerebral cortex expressing pericyte hallmarks can be reprogrammed into neuronal cells by retrovirus-mediated coexpression of the transcription factors Sox2 and Mash1. These induced neuronal cells acquire the ability of repetitive action potential firing and serve as synaptic targets for other neurons, indicating their capability of integrating into neural networks. Genetic fate-mapping in mice expressing an inducible Cre recombinase under the tissue-nonspecific alkaline phosphatase promoter corroborated the pericytic origin of the reprogrammed cells. Our results raise the possibility of functional conversion of endogenous cells in the adult human brain to induced neuronal fates.

Sox2-Mediated Conversion of NG2 Glia into Induced Neurons in the Injured Adult Cerebral Cortex

Summary The adult cerebral cortex lacks the capacity to replace degenerated neurons following traumatic injury. Conversion of nonneuronal cells into induced neurons has been proposed as an innovative strategy toward brain repair. Here, we show that retrovirus-mediated expression of the transcription factors Sox2 and Ascl1, but strikingly also Sox2 alone, can induce the conversion of genetically fate-mapped NG2 glia into induced doublecortin (DCX)+ neurons in the adult mouse cerebral cortex following stab wound injury in vivo. In contrast, lentiviral expression of Sox2 in the unlesioned cortex failed to convert oligodendroglial and astroglial cells into DCX+ cells. Neurons induced following injury mature morphologically and some acquire NeuN while losing DCX. Patch-clamp recording of slices containing Sox2- and/or Ascl1-transduced cells revealed that a substantial fraction of these cells receive synaptic inputs from neurons neighboring the injury site. Thus, NG2 glia represent a potential target for reprogramming strategies toward cortical repair.

Inflammatory changes during epileptogenesis and spontaneous seizures in a mouse model of mesiotemporal lobe epilepsy

Purpose:  Neuroinflammation appears as a prominent feature of the mesiotemporal lobe epilepsy syndrome (MTLE) that is observed in human patients and animal models. However, the precise temporal relationship of its development during epileptogenesis remains to be determined. The aim of the present study was to investigate (1) the time course and spatial distribution of neuronal death associated with seizure development, (2) the time course of microglia and astrocyte activation, and (3) the kinetics of induction of mRNAs from neuroinflammatory‐related proteins during the emergence of recurrent seizures.

Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex

Instructing glial cells to generate neurons may prove to be a strategy to replace neurons that have degenerated. Here, we describe a robust protocol for the efficient in vitro conversion of postnatal astroglia from the mouse cerebral cortex into functional, synapse-forming neurons. This protocol involves two steps: (i) expansion of astroglial cells (7 d) and (ii) astroglia-to-neuron conversion induced by persistent and strong retroviral expression of Neurog2 (encoding neurogenin-2) or Mash1 (also referred to as achaete-scute complex homolog 1 or Ascl1) and/or distal-less homeobox 2 (Dlx2) for generation of glutamatergic or GABAergic neurons, respectively (7–21 d for different degrees of maturity). Our protocol of astroglia-to-neuron conversion by a single neurogenic transcription factor provides a stringent experimental system to study the specification of a selective neuronal subtype, thus offering an alternative to the use of embryonic or neural stem cells. Moreover, it can be a useful model for studies of lineage conversion from non-neuronal cells, with potential for brain regenerative medicine.

Identification and Successful Negotiation of a Metabolic Checkpoint in Direct Neuronal Reprogramming

Despite the widespread interest in direct neuronal reprogramming, the mechanisms underpinning fate conversion remain largely unknown. Our study revealed a critical time point after which cells either successfully convert into neurons or succumb to cell death. Co-transduction with Bcl-2 greatly improved negotiation of this critical point by faster neuronal differentiation. Surprisingly, mutants with reduced or no affinity for Bax demonstrated that Bcl-2 exerts this effect by an apoptosis-independent mechanism. Consistent with a caspase-independent role, ferroptosis inhibitors potently increased neuronal reprogramming by inhibiting lipid peroxidation occurring during fate conversion. Genome-wide expression analysis confirmed that treatments promoting neuronal reprogramming elicit an anti-oxidative stress response. Importantly, co-expression of Bcl-2 and anti-oxidative treatments leads to an unprecedented improvement in glial-to-neuron conversion after traumatic brain injury in vivo, underscoring the relevance of these pathways in cellular reprograming irrespective of cell type in vitro and in vivo.

In vivo reprogramming for tissue repair

Vital organs such as the pancreas and the brain lack the capacity for effective regeneration. To overcome this limitation, an emerging strategy consists of converting resident tissue-specific cells into the cell types that are lost due to disease by a process called in vivo lineage reprogramming. Here we discuss recent breakthroughs in regenerating pancreatic β-cells and neurons from various cell types, and highlight fundamental challenges that need to be overcome for the translation of in vivo lineage reprogramming into therapy.

Reprogramming of Postnatal Astroglia of the Mouse Neocortex into Functional, Synapse-Forming Neurons

Direct conversion of glia into neurons by cellular reprogramming represents a novel approach toward a cell-based therapy of neurodegenerative processes. Here we describe a protocol that allows for the direct and efficient in vitro reprogramming of mouse astroglia from the early postnatal neocortex by forced expression of single neurogenic fate determinants. By selective retrovirus-mediated expression of neurogenin-2 (Neurog2) on the one hand, or the mouse homologue of Distal-less Dlx2 or the mammalian homologue of achaete-schute-1 (Mash1) on the other, it is possible to drive postnatal astroglia in culture toward the genesis of fully functional, synapse-forming, glutamatergic, i.e., excitatory, and GABAergic, i.e., inhibitory, neurons, respectively.

Direct conversion of pericyte-derived cells of the adult human brain into functional neurons

R. Sánchez 1, M. Karow 1, C. Schichor 2, G. Masserdotti 1,3, F. Ortega 1, C. Heinrich 1, S. Gascón 1,3, M.A. Khan 4, D.C. Lie 4, A. Dellavalle 5, G. Cossu 5, R. Goldbrunner 2,6, M. Götz 1,3, B. Berninger 1,3 1 Department of Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, Schillerstrasse 46, D-80336 Munich, Germany 2 Tumor Biology Lab, Neurosurgical Clinic, Klinikum der Universität München, Großhadern, Marchioninistrasse 15, D-81377 Munich, Germany 3 Institute for Stem Cell Research, National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany 4 Research Group/Adult Neural Stem Cells and Neurogenesis, Institute of Developmental Genetics, National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany 5 Division of Regenerative Medicine, San Raffaele Scientific Institute, 58 via Olgettina, Milan 20132, Italy 6 Center for Neurosurgery, University Hospital of Cologne, Kerpener Strasse 62, D-50937 Cologne, Germany Reprogramming of somatic cells into neurons provides a new approach towards cell-based therapy of neurodegenerative diseases. Previous studies have shown that postnatal astroglia from the cerebral cortex of mice can be directly converted into functional neurons in vitro by forced expression of a single neurogenic transcription factor and the synergistic action of 3–4 transcription factors can induce neurogenesis from rodent and human fibroblasts. However, a major challenge for the translation of neuronal reprogramming into therapy concerns the question whether direct conversion of somatic cells into neurons can be achieved from cells residing within the adult human brain. Here we show that cells from the adult human cerebral cortex expressing pericyte hallmarks, such as the PDGF receptor(PDGF ), can be reprogrammed into III-tubulin-, MAP2and NeuN-positive neurons by co-expression of the transcription factors Mash1 (mammalian homologue of achaete-schute-1) and the SRY-related HMG box protein Sox2, but not by either factor alone. These neurons are functional as they acquire the ability of repetitive action potential firing and serve as synaptic targets for other neurons indicating their capability of integrating into neuronal networks. The pericytic origin of these neurons was further corroborated by isolating PDGFR -positive cells from human brain cultures using fluorescence-activated cell sorting and continuous live imaging during reprogramming. Genetic fate-mapping in mice expressing an inducible Cre recombinase under the tissue non-specific alkaline phosphatase promoter confirmed that pericytes from the adult cerebral cortex can be expanded and reprogrammed in vitro into neurons by Sox2 and Mash1. Our results demonstrate that direct neuronal reprogramming can be achieved from somatic cells of adult brain tissue, including of human origin. This data provide strong support for the idea that direct reprogramming of somatic cells endogenous to the adult brain represents a viable approach for cell-based therapies of neurodegenerative diseases.