31 research outputs found
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Frontotemporal dementia causative CHMP2B impairs neuronal endolysosomal traffic-rescue by TMEM106B knockdown.
Mutations in the endosome-associated protein CHMP2B cause frontotemporal dementia and lead to lysosomal storage pathology in neurons. We here report that physiological levels of mutant CHMP2B causes reduced numbers and significantly impaired trafficking of endolysosomes within neuronal dendrites, accompanied by increased dendritic branching. Mechanistically, this is due to the stable incorporation of mutant CHMP2B onto neuronal endolysosomes, which we show renders them unable to traffic within dendrites. This defect is due to the inability of mutant CHMP2B to recruit the ATPase VPS4, which is required for release of CHMP2B from endosomal membranes. Strikingly, both impaired trafficking and the increased dendritic branching were rescued by treatment with antisense oligonucleotides targeting the well validated frontotemporal dementia risk factor TMEM106B, which encodes an endolysosomal protein. This indicates that reducing TMEM106B levels can restore endosomal health in frontotemporal dementia. As TMEM106B is a risk factor for frontotemporal dementia caused by both C9orf72 and progranulin mutations, and antisense oligonucleotides are showing promise as therapeutics for neurodegenerative diseases, our data suggests a potential new strategy for treating the wide range of frontotemporal dementias associated with endolysosomal dysfunction
An Efficient System for Gene Perturbation in Embryonic Hippocampal Progenitors Using Ex Vivo Electroporation Followed by In Vitro Dissociated Cell Culture
We established an efficient cell culture assay that permits combinatorial genetic perturbations in hippocampal progenitors to examine cell-autonomous mechanisms of fate specification. The procedure begins with ex vivo electroporation of isolated, intact embryonic brains, in a manner similar to in utero electroporation but with greatly improved access and targeting. The electroporated region is then dissected and transiently maintained in organotypic explant culture, followed by dissociation and plating of cells on coverslips for in vitro culture. This assay recapitulates data obtained in vivo with respect to the neuron-glia cell fate switch and can be effectively used to test intrinsic or extrinsic factors that regulate this process. The advantages of this ex vivo procedure over in utero electroporation include the fact that distinct combinations of perturbative reagents can be introduced in different embryos from a single litter, and issues related to embryonic lethality of transgenic animals can be circumvented
A minimal element in 5′UTR of insulin mRNA mediates its translational regulation by glucose
Lhx2 regulates a cortex-specific mechanism for barrel formation.
LIM homeodomain transcription factors are critical regulators of early development in multiple systems but have yet to be examined for a role in circuit formation. The LIM homeobox gene Lhx2 is expressed in cortical progenitors during development and also in the superficial layers of the neocortex in maturity. However, analysis of Lhx2 function at later stages of cortical development has been hampered by severe phenotypes associated with early loss of function. We identified a particular Cre-recombinase line that acts in the cortical primordium after its specification is complete, permitting an analysis of Lhx2 function in neocortical lamination, regionalization, and circuit formation by selective elimination of Lhx2 in the dorsal telencephalon. We report a profound disruption of cortical neuroanatomical and molecular features upon loss of Lhx2 in the cortex from embryonic day 11.5. A unique feature of cortical circuitry, the somatosensory barrels, is undetectable, and molecular patterning of cortical regions appears disrupted. Surprisingly, thalamocortical afferents innervate the mutant cortex with apparently normal regional specificity. Electrophysiological recordings reveal a loss of responses evoked by stimulation of individual whiskers, but responses to simultaneous stimulation of multiple whiskers were present, suggesting that thalamic afferents are unable to organize the neurocircuitry for barrel formation because of a cortex-specific requirement of Lhx2. We report that Lhx2 is required for the expression of transcription factor paired box gene 6, axon guidance molecule Ephrin A5, and the receptor NMDA receptor 1. These genes may mediate Lhx2 function in the formation of specialized neurocircuitry necessary for neocortical function
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Lhx2 regulates a cortex-specific mechanism for barrel formation.
LIM homeodomain transcription factors are critical regulators of early development in multiple systems but have yet to be examined for a role in circuit formation. The LIM homeobox gene Lhx2 is expressed in cortical progenitors during development and also in the superficial layers of the neocortex in maturity. However, analysis of Lhx2 function at later stages of cortical development has been hampered by severe phenotypes associated with early loss of function. We identified a particular Cre-recombinase line that acts in the cortical primordium after its specification is complete, permitting an analysis of Lhx2 function in neocortical lamination, regionalization, and circuit formation by selective elimination of Lhx2 in the dorsal telencephalon. We report a profound disruption of cortical neuroanatomical and molecular features upon loss of Lhx2 in the cortex from embryonic day 11.5. A unique feature of cortical circuitry, the somatosensory barrels, is undetectable, and molecular patterning of cortical regions appears disrupted. Surprisingly, thalamocortical afferents innervate the mutant cortex with apparently normal regional specificity. Electrophysiological recordings reveal a loss of responses evoked by stimulation of individual whiskers, but responses to simultaneous stimulation of multiple whiskers were present, suggesting that thalamic afferents are unable to organize the neurocircuitry for barrel formation because of a cortex-specific requirement of Lhx2. We report that Lhx2 is required for the expression of transcription factor paired box gene 6, axon guidance molecule Ephrin A5, and the receptor NMDA receptor 1. These genes may mediate Lhx2 function in the formation of specialized neurocircuitry necessary for neocortical function
Dmrt5, a novel neurogenic factor, reciprocally regulates Lhx2 to control the neuron-glia cell fate switch in the developing hippocampus
Regulation of the neuron-glia cell-fate switch is a critical step in the development of the CNS. Previously, we demonstrated that Lhx2 is a necessary and sufficient regulator of this process in the mouse hippocampal primordium, such that Lhx2 overexpression promotes neurogenesis and suppresses gliogenesis, whereas loss of Lhx2 has the opposite effect.Wetested a series of transcription factors for their ability to mimic Lhx2 overexpression and suppress baseline gliogenesis, and also to compensate for loss of Lhx2 and suppress the resulting enhanced level of gliogenesis in the hippocampus. Here, we demonstrate a novel function of Dmrt5/Dmrta2 as a neurogenic factor in the developing hippocampus. We show that Dmrt5, as well as known neurogenic factors Neurog2 and Pax6, can each not only mimic Lhx2 overexpression, but also can compensate for loss of Lhx2 to different extents. We further uncover a reciprocal regulatory relationship between Dmrt5 and Lhx2, such that each can compensate for loss of the other. Dmrt5 and Lhx2 also have opposing regulatory control on Pax6 and Neurog2, indicating a complex bidirectionally regulated network that controls the neuron-glia cell-fate switch. Finally, we confirm that Lhx2 binds a highly conserved putative enhancer of Dmrt5, suggesting an evolutionarily conserved regulatory relationship between these factors. Our findings uncover a complex network that involves Lhx2, Dmrt5, Neurog2, and Pax6, and that ensures the appropriate amount and timing of neurogenesis and gliogenesis in the developing hippocampus.Significance Statement Weidentify Dmrt5 as a novel regulator of the neuronglia cell-fate switch in the developing hippocampus.Wedemonstrate Dmrt5 to be neurogenic, and reciprocally regulated by Lhx2: loss of either factor promotes gliogenesis; overexpression of either factor suppresses gliogenesis and promotes neurogenesis; each can substitute for loss of the other. Furthermore, each factor has opposing effects on established neurogenic genes Neurog2 and Pax6. Dmrt5 is known to suppress their expression, and we show that Lhx2 is required to maintain it. Our study reveals a complex regulatory network with bidirectional control of a fundamental feature of CNS development, the control of the production of neurons versus astroglia in the developing hippocampus.SCOPUS: ar.jinfo:eu-repo/semantics/publishe
Gene Regulatory Networks modulated by LHX2 in the Ncp and Hcp.
(A, C) Venn diagrams depicting the number of genes occupied by LHX2 and dysregulated (blue: downregulated, red: upregulated) upon loss of Lhx2 in the Ncp (A) and Hcp (C) respectively to identify direct targets of LHX2 in the Ncp and Hcp. (B, D) Genes dysregulated upon loss of Lhx2 in the Ncp (B) and Hcp (D) respectively, categorized by “Direct” or (direct + indirect) = “All” targets, mapped to the cell-type specific gene enrichment profiles in [28]) to identify progenitor-enriched (grey) and neuron-enriched genes (black). (E) Venn diagram comparing the direct targets of LHX2 that are dysregulated upon loss of Lhx2 in the Ncp (112 downregulated; 118 upregulated) and Hcp (70 downregulated; 153 upregulated), and in both tissues (43 downregulated; 35 upregulated). (F) Comparison of LHX2 occupancy in the E10.5 dorsal telencephalon (dtel; blue circle) with that in the E12.5 Ncp (red) and Hcp (green) results in genes occupied in all these three tissues (716, yellow), in the E10.5 dtel and the E12.5 Ncp (286, red) or the E12.5 Hcp (1252, green). (G) Venn diagram comparing the genes in E (LHX2 direct targets) that are also occupied by LHX2 at E10.5. In the Ncp, there are 62 downregulated 59 upregulated genes. In the Hcp there are 33 downregulated upregulated 94 upregulated. 37 downregulated and 25 upregulated are common to both tissues. (H) Heatmaps displaying genes occupied by LHX2. Cluster 1: Occupancy at both E10.5 (dtel) and E12.5 (Ncp and Hcp). Cluster 2: Occupancy at only E10.5. (I-L) KEGG pathway analysis (GO: BP) of genes identified in (E, G) reveals 4 pathways dysregulated upon loss of Lhx2 in the Ncp (red bars) and Hcp (green bars). Individual fold changes are plotted from the RNA-seq data (black: genes occupied by LHX2 at E12.5 and E10.5; blue: occupied only at E12.5).</p
LHX2 occupancy in the E12.5 mouse neocortical and hippocampal primordia (Ncp and Hcp, respectively).
(A) Schematic representation of the E12.5 mouse brain. (B) Progenitor markers PAX6, LHX2, and TBR2 immunostaining/in situ hybridization (Lhx2) in the Ncp and Hcp. (C-G) LHX2 ChIP-seq data in the Ncp and Hcp. Plots of PePr peak-called regions in the Ncp and Hcp show TF LHX2 occupancy in each tissue. Only statistically significant peaks were used for further analysis (p-value 0.0001 and fold change over input: cut off >10 fold) (C); The number of LHX2 occupancy peaks and associated number of genes (D); common genes occupied by LHX2 between the Ncp and Hcp (E); Percentage of LHX2 occupancy peaks categorized by type of genomic region (F); LHX2-occupied genes enriched in different cortical cell types identified by gene enrichment profiles in [28](G). The scale bars in B are 100 ÎĽm.</p
Worksheet containing raw data of DARs identified in Fig 2B, a list of annotated DARs identified in Fig 2B, a comparative analysis of genes associated with DARs and gene expression in wtHcp vs wtNcp related to S1B Fig.
Worksheet containing raw data of DARs identified in Fig 2B, a list of annotated DARs identified in Fig 2B, a comparative analysis of genes associated with DARs and gene expression in wtHcp vs wtNcp related to S1B Fig.</p