The mammalian nervous system comprises many distinct neuronal subtypes, each with its own phenotype and differential sensitivity to degenerative disease. Although specific neuronal types can be ...isolated from rodent embryos or engineered from stem cells for translational studies, transcription factor-mediated reprogramming might provide a more direct route to their generation. Here we report that the forced expression of select transcription factors is sufficient to convert mouse and human fibroblasts into induced motor neurons (iMNs). iMNs displayed a morphology, gene expression signature, electrophysiology, synaptic functionality, in vivo engraftment capacity, and sensitivity to degenerative stimuli similar to those of embryo-derived motor neurons. We show that the converting fibroblasts do not transit through a proliferative neural progenitor state, and thus form bona fide motor neurons via a route distinct from embryonic development. Our findings demonstrate that fibroblasts can be converted directly into a specific differentiated and functional neural subtype, the spinal motor neuron.
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► Human and mouse fibroblasts are directly converted to induced motor neurons (iMNs) ► iMNs are molecularly and electrophysiologically bona fide motor neurons ► iMNs innervate muscle, integrate into CNS in vivo, and recapitulate ALS phenotypes ► A Nestin+ progenitor state is bypassed during transdifferentiation into iMNs
Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are ...translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases.
Frontotemporal dementia (FTD) because of MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem ...cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations because of the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, glutamatergic signaling pathways, and regulators, including the RNA-binding protein ELAVL4, and increased stress granules. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function, and build-up of tau and P-tau-S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons as seen in individuals with FTD. Mutant neurons are susceptible to glutamate toxicity, which can be rescued pharmacologically by the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede neurodegeneration, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.
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•Tau and P-tau accumulation and autophagy disruption in tau-V337M organoids•Accelerated synaptic maturation and loss of glutamatergic cortical-layer neurons•Altered ELAVL4 expression, dysregulated splicing, accelerated synaptic maturation•Rescue of susceptibility to glutamatergic toxicity by PIKFYVE inhibitor apilimod
Characterization of iPSC-derived cerebral organoids with the tau-V337M mutation, which causes frontotemporal dementia, reveals changes preceding neuron death as potential targets for therapeutic intervention, as demonstrated by rescue of susceptibility to glutamatergic toxicity by the PIKFYVE inhibitor apilimod.
The groundbreaking technologies of induced pluripotency and lineage conversion have generated a genuine opportunity to address fundamental aspects of the diseases that affect the nervous system. ...These approaches have granted us unrestricted access to the brain and spinal cord of patients and have allowed for the study of disease in the context of human cells, expressing physiological levels of proteins and under each patient's unique genetic constellation. Along with this unprecedented opportunity have come significant challenges, particularly in relation to patient variability, experimental design and data interpretation. Nevertheless, significant progress has been achieved over the past few years both in our ability to create the various neural subtypes that comprise the nervous system and in our efforts to develop cellular models of disease that recapitulate clinical findings identified in patients. In this Review, we present tables listing the various human neural cell types that can be generated and the neurological disease modeling studies that have been reported, describe the current state of the field, highlight important breakthroughs and discuss the next steps and future challenges.
Kiskinis & Ichida highlight recent progress in neurological disease modeling using iPSCs. The reconstitution of the three‐dimensional architecture, including ageing or metabolic parameters will inform the complex nature of neurologic conditions.
Abstract Recent landmark studies have demonstrated the production of disease-relevant human cell types by two different methods; differentiation of stem cells using external morphogens or lineage ...conversion using genetic factors. Directed differentiation changes embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into a desired cell type by providing developmental cues in an in vitro environment. Direct reprogramming is achieved by the introduction of exogenous lineage specific transcription factors to convert any somatic cell type into another, thereby bypassing an intermediate pluripotent stage. A variety of somatic cell types such as blood, keratinocytes and fibroblasts can be used to derive iPSC cells. However, the process is time consuming,laborious, expensive and gives rise to cells with reported epigenetic heterogeneity even amongst different iPSC lines from same patient which could propagate phenotypic variability. A major concern with the use of pluripotent cells as starting material for cell replacement therapy is their incomplete differentiation and their propensity to form tumors following transplantation. In comparison, transcription factor mediated reprogramming offers a direct route to target cell types. This could allow for rapid comparison of large cohorts of patient and control samples at a given time for disease modeling. Additionally, transcription factors that drive maturation may yield more functionally mature cells than directed differentiation. Several studies have demonstrated the feasibility of generating of cell types such as cardiomyocytes, hepatocytes, and neurons from fibroblasts. Here, we will discuss recent advances and key challenges regarding direct reprogramming of somatic cell types into diverse neural cells. This article is part of a Special Issue entitled SI: Exploiting human neurons.
Nucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. Unconventional translation (RAN translation) of C9orf72 ...repeats generates dipeptide repeat proteins that can cause neurodegeneration. We performed a genetic screen for regulators of RAN translation and identified small ribosomal protein subunit 25 (RPS25), presenting a potential therapeutic target for C9orf72-related amyotrophic lateral sclerosis and frontotemporal dementia and other neurodegenerative diseases caused by nucleotide repeat expansions.
Direct lineage conversion offers a fast and simple method to study mature neurons in vitro, but its utility for investigating neurodevelopment has remained unclear. In this issue of Cell Stem Cell, ...Chanda et al. (2019) use Ngn2-induced neurons to elucidate the pathogenic mechanisms of the teratogenic compound valproic acid.
Direct lineage conversion offers a fast and simple method to study mature neurons in vitro, but its utility for investigating neurodevelopment has remained unclear. In this issue of Cell Stem Cell, Chanda et al. (2019) use Ngn2-induced neurons to elucidate the pathogenic mechanisms of the teratogenic compound valproic acid.
•G4C2 hexanucleotide repeat expansions in C9ORF72 lead to protein aggregation and nuclear stress that contribute to ALS.•Loss-of-function effects of C9orf72 are marked by impaired proteostasis, ...immune cell dysfunction and autoimmunity.•Autophagic dysfunction may decrease immune self-tolerance leading to autoimmunity that either initiates or exacerbates ALS.
Amyotrophic lateral sclerosis (ALS) is a rapidly progressing disease that affects upper and lower motor neurons eventually leading to paralysis and death by respiratory dysfunction. The most common genetic variant among ALS patients is a hexanucleotide repeat expansion within the first intron of the gene C9ORF72. This expansion elicits a complex cascade of events as a result of both gain- and loss-of-function mechanisms that contribute to neurodegeneration. Increasing evidence suggests that this repeat expansion in C9ORF72 also influences the immune homeostasis. In this review, we consolidate the current understanding of C9ORF72-mediated pathogenesis in both the central nervous system and peripheral immune system and propose mechanisms by which the immune system contributes to ALS.
Repairing the blood-brain barrier McMahon, Andrew P; Ichida, Justin K
Science (American Association for the Advancement of Science),
02/2022, Letnik:
375, Številka:
6582
Journal Article
Direct lineage conversion could provide a rich source of somatic cell types for translational medicine, but concerns over the use of transgenic reprogramming factors have limited its potential. In ...this issue of Cell Stem Cell, Li et al. (2015) and Hu et al. (2015) identify small-molecule cocktails that can convert fibroblasts into functional neurons without exogenous genetic factors.
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