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Open accessJournal ArticleDOI: 10.1016/J.STEM.2020.10.015

CRISPR-Mediated Induction of Neuron-Enriched Mitochondrial Proteins Boosts Direct Glia-to-Neuron Conversion.

04 Mar 2021-Cell Stem Cell (Elsevier)-Vol. 28, Iss: 3, pp 584-584
Abstract: Summary Astrocyte-to-neuron conversion is a promising avenue for neuronal replacement therapy. Neurons are particularly dependent on mitochondrial function, but how well mitochondria adapt to the new fate is unknown. Here, we determined the comprehensive mitochondrial proteome of cortical astrocytes and neurons, identifying about 150 significantly enriched mitochondrial proteins for each cell type, including transporters, metabolic enzymes, and cell-type-specific antioxidants. Monitoring their transition during reprogramming revealed late and only partial adaptation to the neuronal identity. Early dCas9-mediated activation of genes encoding mitochondrial proteins significantly improved conversion efficiency, particularly for neuron-enriched but not astrocyte-enriched antioxidant proteins. For example, Sod1 not only improves the survival of the converted neurons but also elicits a faster conversion pace, indicating that mitochondrial proteins act as enablers and drivers in this process. Transcriptional engineering of mitochondrial proteins with other functions improved reprogramming as well, demonstrating a broader role of mitochondrial proteins during fate conversion.

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Topics: Mitochondrion (57%), Proteome (51%)
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Open access
01 Jan 2009-

1,353 Citations


Open accessJournal ArticleDOI: 10.3389/FNMOL.2021.642016
Ana-Maria Oproescu1, Sisu Han2, Sisu Han1, Carol Schuurmans2  +1 moreInstitutions (2)
Abstract: Historically, the mammalian brain was thought to lack stem cells as no new neurons were found to be made in adulthood. That dogma changed ∼25 years ago with the identification of neural stem cells (NSCs) in the adult rodent forebrain. However, unlike rapidly self-renewing mature tissues (e.g., blood, intestinal crypts, skin), the majority of adult NSCs are quiescent, and those that become 'activated' are restricted to a few neurogenic zones that repopulate specific brain regions. Conversely, embryonic NSCs are actively proliferating and neurogenic. Investigations into the molecular control of the quiescence-to-proliferation-to-differentiation continuum in the embryonic and adult brain have identified proneural genes encoding basic-helix-loop-helix (bHLH) transcription factors (TFs) as critical regulators. These bHLH TFs initiate genetic programs that remove NSCs from quiescence and drive daughter neural progenitor cells (NPCs) to differentiate into specific neural cell subtypes, thereby contributing to the enormous cellular diversity of the adult brain. However, new insights have revealed that proneural gene activities are context-dependent and tightly regulated. Here we review how proneural bHLH TFs are regulated, with a focus on the murine cerebral cortex, drawing parallels where appropriate to other organisms and neural tissues. We discuss upstream regulatory events, post-translational modifications (phosphorylation, ubiquitinylation), protein-protein interactions, epigenetic and metabolic mechanisms that govern bHLH TF expression, stability, localization, and consequent transactivation of downstream target genes. These tight regulatory controls help to explain paradoxical findings of changes to bHLH activity in different cellular contexts.

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Topics: Proneural genes (63%), Neural stem cell (55%), ASCL1 (54%) ... read more

3 Citations


Open accessJournal ArticleDOI: 10.1016/J.CONB.2021.05.003
Ryohei Iwata1, Pierre Vanderhaeghen1Institutions (1)
Abstract: Neural stem cells (NSCs) undergo massive molecular and cellular changes during neuronal differentiation. These include mitochondria and metabolism remodelling, which were thought to be mostly permissive cues, but recent work indicates that they are causally linked to neurogenesis. Striking remodelling of mitochondria occurs right after mitosis of NSCs, which influences the postmitotic daughter cells towards self-renewal or differentiation. The transitioning to neuronal fate requires metabolic rewiring including increased oxidative phosphorylation activity, which drives transcriptional and epigenetic effects to influence cell fate. Mitochondria metabolic pathways also contribute in an essential way to the regulation of NSC proliferation and self-renewal. The influence of mitochondria and metabolism on neurogenesis is conserved from fly to human systems, but also displays striking differences linked to cell context or species. These new findings have important implications for our understanding of neurodevelopmental diseases and possibly human brain evolution.

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Topics: Neurogenesis (59%), Neural stem cell (53%), Cell fate determination (52%)

2 Citations


Open accessJournal ArticleDOI: 10.1016/J.CONB.2021.03.014
Abstract: Regenerative approaches have made such a great progress, now aiming toward replacing the exact neurons lost upon injury or neurodegeneration. Transplantation and direct reprogramming approaches benefit from identification of molecular programs for neuronal subtype specification, allowing engineering of more precise neuronal subtypes. Disentangling subtype diversity from dynamic transcriptional states presents a challenge now. Adequate identity and connectivity is a prerequisite to restore neuronal network function, which is achieved by transplanted neurons generating the correct output and input, depending on the location and injury condition. Direct neuronal reprogramming of local glial cells has also made great progress in achieving high efficiency of conversion, with adequate output connectivity now aiming toward the goal of replacing neurons in a noninvasive approach.

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Topics: Transplantation (52%)

2 Citations


Open accessJournal ArticleDOI: 10.1016/J.CELREP.2021.109409
J. Kempf1, K. Knelles1, B.A. Hersbach1, David Petrik2  +9 moreInstitutions (2)
20 Jul 2021-Cell Reports
Abstract: Astrocytes are a viable source for generating new neurons via direct conversion. However, little is known about the neurogenic cascades triggered in astrocytes from different regions of the CNS. Here, we examine the transcriptome induced by the proneural factors Ascl1 and Neurog2 in spinal cord-derived astrocytes in vitro. Each factor initially elicits different neurogenic programs that later converge to a V2 interneuron-like state. Intriguingly, patch sequencing (patch-seq) shows no overall correlation between functional properties and the transcriptome of the heterogenous induced neurons, except for K-channels. For example, some neurons with fully mature electrophysiological properties still express astrocyte genes, thus calling for careful molecular and functional analysis. Comparing the transcriptomes of spinal cord- and cerebral-cortex-derived astrocytes reveals profound differences, including developmental patterning cues maintained in vitro. These relate to the distinct neuronal identity elicited by Ascl1 and Neurog2 reflecting their developmental functions in subtype specification of the respective CNS region.

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Topics: Astrocyte (53%), ASCL1 (50%)

1 Citations


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67 results found


Journal ArticleDOI: 10.1038/362059A0
Daniel R. Rosen1Institutions (1)
04 Mar 1993-Nature
Abstract: Amyotrophic lateral sclerosis (ALS) is a degenerative disorder of motor neurons in the cortex, brainstem and spinal cord. Its cause is unknown and it is uniformly fatal, typically within five years. About 10% of cases are inherited as an autosomal dominant trait, with high penetrance after the sixth decade. In most instances, sporadic and autosomal dominant familial ALS (FALS) are clinically similar. We have previously shown that in some but not all FALS pedigrees the disease is linked to a genetic defect on chromosome 21q (refs 8, 9). Here we report tight genetic linkage between FALS and a gene that encodes a cytosolic, Cu/Zn-binding superoxide dismutase (SOD1), a homodimeric metalloenzyme that catalyzes the dismutation of the toxic superoxide anion O2.- to O2 and H2O2 (ref. 10). Given this linkage and the potential role of free radical toxicity in other neurodenegerative disorders, we investigated SOD1 as a candidate gene in FALS. We identified 11 different SOD1 missense mutations in 13 different FALS families.

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Topics: Copper chaperone for superoxide dismutase (60%), SOD1 (59%), Autosomal dominant trait (59%) ... read more

6,370 Citations


Journal ArticleDOI: 10.1038/NMETH.1322
01 May 2009-Nature Methods
Abstract: A method, filter-aided sample preparation (FASP) combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics, allowing deeper proteomic coverage in a shorter analysis time, using small sample amounts. We describe a method, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics. We completely solubilized the proteome in sodium dodecyl sulfate, which we then exchanged by urea on a standard filtration device. Peptides eluted after digestion on the filter were pure, allowing single-run analyses of organelles and an unprecedented depth of proteome coverage.

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Topics: Proteome (51%), Sample preparation (51%)

4,899 Citations


Open accessJournal ArticleDOI: 10.1038/NATURE14136
29 Jan 2015-Nature
Abstract: Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.

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Topics: Cas9 (55%), Regulation of gene expression (54%), Gene (54%) ... read more

1,769 Citations


Open accessJournal ArticleDOI: 10.1016/J.CELL.2008.06.016
David J. Pagliarini1, Sarah E. Calvo, Betty Chang2, Sunil A Sheth  +15 moreInstitutions (5)
11 Jul 2008-Cell
Abstract: Mitochondria are complex organelles whose dysfunction underlies a broad spectrum of human diseases. Identifying all of the proteins resident in this organelle and understanding how they integrate into pathways represent major challenges in cell biology. Toward this goal, we performed mass spectrometry, GFP tagging, and machine learning to create a mitochondrial compendium of 1098 genes and their protein expression across 14 mouse tissues. We link poorly characterized proteins in this inventory to known mitochondrial pathways by virtue of shared evolutionary history. Using this approach, we predict 19 proteins to be important for the function of complex I (CI) of the electron transport chain. We validate a subset of these predictions using RNAi, including C8orf38, which we further show harbors an inherited mutation in a lethal, infantile CI deficiency. Our results have important implications for understanding CI function and pathogenesis and, more generally, illustrate how our compendium can serve as a foundation for systematic investigations of mitochondria.

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1,682 Citations



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