The role of chromatin associated protein HMGB2 in setting up permissive chromatin states for direct glia to neuron conversion

The adult mammalian brain has a limited capacity to replace the loss of neurons following the traumatic brain injury or disease, therefore requiring innovative strategies to promote tissue regeneration and functional repair of the central nervous system (CNS). Direct lineage reprogramming of non-neuronal cell types (reactive astrocytes and oligodendrocytes), resident within the injured CNS to generate new neurons is a promising approach to repair damaged brain. Despite very good conversion rate of isolated postnatal astrocytes in vitro and recent advances to produce induced neuronal cells in vivo, the molecular understanding of the reprogramming process remains largely unknown. Toward this end, we developed an in vitro model with adjusted growth factor composition lacking epidermal growth factor (EGF) that reduces the reprogramming efficiency of astroglia into neurons to the rate similar to reactive astrocytes in vivo. By comparing the cultures prone and resistant to reprogramming we aimed to identify molecular features required for the efficient conversion. The proteome analysis of EGF+FGF and FGF cultures revealed the chromatin state changes as the main factor that could maintain the astrocytes in the glial lineage even after neurogenic factor overexpression. To test this hypothesis, we overexpressed the most regulated chromatin-related protein, HMGB2, together with NEUROG2 and analysed the reprogramming efficiency. Indeed, the simultaneous overexpression of both factors in the astrocytes resistant to the fate transition significantly increased direct reprogramming suggesting the role of chromatin-associated proteins in bypassing the lineage roadblocks. To understand the role of global chromatin rearrangement, we performed ATAC-sequencing comparing both culture conditions and searched for HMGB2 induced changes in the chromatin compaction. The chromatin accessibility analysis revealed an enrichment of opened promoters assigned to the neuronal genes in the astrocytes with good reprogramming capacity and the ability of HMGB2 to open these specific promoters in the culture resistant to reprogramming. In order to access the transcriptional changes induced by HMGB2 and NEUROG2 overexpression, we performed RNA-sequencing that indicated the involvement of HMGB2 in repression of the myogenic alternative fate, possibly by the opening the repressor binding sites. Taken together, we identified the novel candidate HMGB2 in the prospect of direct neuronal reprogramming making the chromatin states in the glial cells permissive for lineage conversion and thereby enabling acquisition of the neuronal phenotype in the cultures with limited reprogramming capacity

The role of chromatin associated protein HMGB2 in setting up permissive chromatin states for direct glia to neuron conversion

The adult mammalian brain has a limited capacity to replace the loss of neurons following the traumatic brain injury or disease, therefore requiring innovative strategies to promote tissue regeneration and functional repair of the central nervous system (CNS). Direct lineage reprogramming of non-neuronal cell types (reactive astrocytes and oligodendrocytes), resident within the injured CNS to generate new neurons is a promising approach to repair damaged brain. Despite very good conversion rate of isolated postnatal astrocytes in vitro and recent advances to produce induced neuronal cells in vivo, the molecular understanding of the reprogramming process remains largely unknown. Toward this end, we developed an in vitro model with adjusted growth factor composition lacking epidermal growth factor (EGF) that reduces the reprogramming efficiency of astroglia into neurons to the rate similar to reactive astrocytes in vivo. By comparing the cultures prone and resistant to reprogramming we aimed to identify molecular features required for the efficient conversion. The proteome analysis of EGF+FGF and FGF cultures revealed the chromatin state changes as the main factor that could maintain the astrocytes in the glial lineage even after neurogenic factor overexpression. To test this hypothesis, we overexpressed the most regulated chromatin-related protein, HMGB2, together with NEUROG2 and analysed the reprogramming efficiency. Indeed, the simultaneous overexpression of both factors in the astrocytes resistant to the fate transition significantly increased direct reprogramming suggesting the role of chromatin-associated proteins in bypassing the lineage roadblocks. To understand the role of global chromatin rearrangement, we performed ATAC-sequencing comparing both culture conditions and searched for HMGB2 induced changes in the chromatin compaction. The chromatin accessibility analysis revealed an enrichment of opened promoters assigned to the neuronal genes in the astrocytes with good reprogramming capacity and the ability of HMGB2 to open these specific promoters in the culture resistant to reprogramming. In order to access the transcriptional changes induced by HMGB2 and NEUROG2 overexpression, we performed RNA-sequencing that indicated the involvement of HMGB2 in repression of the myogenic alternative fate, possibly by the opening the repressor binding sites. Taken together, we identified the novel candidate HMGB2 in the prospect of direct neuronal reprogramming making the chromatin states in the glial cells permissive for lineage conversion and thereby enabling acquisition of the neuronal phenotype in the cultures with limited reprogramming capacity

Changes in the proliferative program limit astrocyte homeostasis in the aged post-traumatic murine cerebral cortex

Aging leads to adverse outcomes after traumatic brain injury. The mechanisms underlying these defects, however, are not yet clear. In this study, we found that astrocytes in the aged post-traumatic cerebral cortex develop a significantly reduced proliferative response, resulting in reduced astrocyte numbers in the penumbra. Moreover, experiments of reactive astrocytes in vitro reveal that their diminished proliferation is due to an age-related switch in the division mode with reduced cell-cycle re-entry rather than changes in cell-cycle length. Notably, reactive astrocytes in vivo and in vitro become refractory to stimuli increasing their proliferation during aging, such as Sonic hedgehog signaling. These data demonstrate for the first time that age-dependent, most likely intrinsic changes in the proliferative program of reactive astrocytes result in their severely hampered proliferative response to traumatic injury thereby affecting astrocyte homeostasis.Instituto de Biotecnologia y Biologia Molecula

Changes in the proliferative program limit astrocyte homeostasis in the aged post-traumatic murine cerebral cortex

Aging leads to adverse outcomes after traumatic brain injury. The mechanisms underlying these defects, however, are not yet clear. In this study, we found that astrocytes in the aged post-traumatic cerebral cortex develop a significantly reduced proliferative response, resulting in reduced astrocyte numbers in the penumbra. Moreover, experiments of reactive astrocytes in vitro reveal that their diminished proliferation is due to an age-related switch in the division mode with reduced cell-cycle re-entry rather than changes in cell-cycle length. Notably, reactive astrocytes in vivo and in vitro become refractory to stimuli increasing their proliferation during aging, such as Sonic hedgehog signaling. These data demonstrate for the first time that age-dependent, most likely intrinsic changes in the proliferative program of reactive astrocytes result in their severely hampered proliferative response to traumatic injury thereby affecting astrocyte homeostasis.Instituto de Biotecnologia y Biologia Molecula

Innate Immune Pathways Promote Oligodendrocyte Progenitor Cell Recruitment to the Injury Site in Adult Zebrafish Brain

The oligodendrocyte progenitors (OPCs) are at the front of the glial reaction to the traumatic brain injury. However, regulatory pathways steering the OPC reaction as well as the role of reactive OPCs remain largely unknown. Here, we compared a long-lasting, exacerbated reaction of OPCs to the adult zebrafish brain injury with a timely restricted OPC activation to identify the specific molecular mechanisms regulating OPC reactivity and their contribution to regeneration. We demonstrated that the influx of the cerebrospinal fluid into the brain parenchyma after injury simultaneously activates the toll-like receptor 2 (Tlr2) and the chemokine receptor 3 (Cxcr3) innate immunity pathways, leading to increased OPC proliferation and thereby exacerbated glial reactivity. These pathways were critical for long-lasting OPC accumulation even after the ablation of microglia and infiltrating monocytes. Importantly, interference with the Tlr1/2 and Cxcr3 pathways after injury alleviated reactive gliosis, increased new neuron recruitment, and improved tissue restoration

Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition

RNA-binding proteins (RBPs) and long non-coding RNAs (lncRNAs) are key regulators of gene expression, but their joint functions in coordinating cell fate decisions are poorly understood. Here we show that the expression and activity of the RBP TDP-43 and the long isoform of the lncRNA Neat1, the scaffold of the nuclear compartment "paraspeckles," are reciprocal in pluripotent and differentiated cells because of their cross-regulation. In pluripotent cells, TDP-43 represses the formation of paraspeckles by enhancing the polyadenylated short isoform of Neat1. TDP-43 also promotes pluripotency by regulating alternative polyadenylation of transcripts encoding pluripotency factors, including Sox2, which partially protects its 3' UTR from miR-21-mediated degradation. Conversely, paraspeckles sequester TDP-43 and other RBPs from mRNAs and promote exit from pluripotency and embryonic patterning in the mouse. We demonstrate that cross-regulation between TDP-43 and Neat1 is essential for their efficient regulation of a broad network of genes and, therefore, of pluripotency and differentiation

Changes in the proliferative program limit astrocyte Hhmeostasis in the aged post-traumatic murine cerebral cortex.

Aging leads to adverse outcomes after traumatic brain injury. The mechanisms underlying these defects, however, are not yet clear. In this study, we found that astrocytes in the aged post-traumatic cerebral cortex develop a significantly reduced proliferative response, resulting in reduced astrocyte numbers in the penumbra. Moreover, experiments of reactive astrocytes in vitro reveal that their diminished proliferation is due to an age-related switch in the division mode with reduced cell-cycle re-entry rather than changes in cell-cycle length. Notably, reactive astrocytes in vivo and in vitro become refractory to stimuli increasing their proliferation during aging, such as Sonic hedgehog signaling. These data demonstrate for the first time that age-dependent, most likely intrinsic changes in the proliferative program of reactive astrocytes result in their severely hampered proliferative response to traumatic injury thereby affecting astrocyte homeostasis