The central nervous system is particularly susceptible to DNA repair deficiency, which renders a variety of neurodevelopmental and neurodegenerative disorders in humans. It is generally believed that ...DNA damage occurs upon repetitive replication and oxidative stress in highly proliferating neuroprogenitor cells (NPs), or due to high rates of metabolism and active neuronal activity in terminally differentiated neurons. DNA double‐stranded breaks (DSBs) and single‐stranded breaks (SSBs) constitute the most prevalent forms of DNA damage, which can result in neuronal apoptosis if unrepaired. Despite these notions, there are still gaps in our knowledge regarding the mechanism and specificity of DNA damage and repair in the neural development and the homeostasis of neural tissues. Recent studies have identified recurrent DSBs within neuronal long genes in NPs and ‘programmed’ SSBs in neuronal activity genes. However, the physiological function of these DNA breakages in the nervous system has not been so far explored. In this review, we summarise the recent advances in the field of DNA damage and DNA repair in neural development and neuropathies.
Programmed DNA breaks occur in neural progenitors during gene rearrangement. In neuronal cells, intrinsic and extrinsic signals can induce DNA breaks in promoters of response genes. Under DNA repair deficient conditions, these DNA damages accumulate and represent an etiological factor for human neurodevelopmental and neurodegenerative pathologies. Here, we summarise recent research on ‘programmed’ DNA breaks and their repair in these neural cells and imply their physiological functions in the nervous system.
Modeling Parkinson's disease (PD) using advanced experimental in vitro models is a powerful tool to study disease mechanisms and to elucidate unexplored aspects of this neurodegenerative disorder. ...Here, we demonstrate that three-dimensional (3D) differentiation of expandable midbrain floor plate neural progenitor cells (mfNPCs) leads to organoids that resemble key features of the human midbrain. These organoids are composed of midbrain dopaminergic neurons (mDANs), which produce and secrete dopamine. Midbrain-specific organoids derived from PD patients carrying the
G2019S mutation recapitulate disease-relevant phenotypes. Automated high-content image analysis shows a decrease in the number and complexity of mDANs in
G2019S compared to control organoids. The floor plate marker FOXA2, required for mDAN generation, increases in PD patient-derived midbrain organoids, suggesting a neurodevelopmental defect in mDANs expressing
G2019S. Thus, we provide a robust method to reproducibly generate 3D human midbrain organoids containing mDANs to investigate PD-relevant patho-mechanisms.
Epithelial-to-mesenchymal transition (EMT) is a developmental process important for cell fate determination. Fibroblasts, a product of EMT, can be reset into induced pluripotent stem cells (iPSCs) ...via exogenous transcription factors but the underlying mechanism is unclear. Here we show that the generation of iPSCs from mouse fibroblasts requires a mesenchymal-to-epithelial transition (MET) orchestrated by suppressing pro-EMT signals from the culture medium and activating an epithelial program inside the cells. At the transcriptional level, Sox2/Oct4 suppress the EMT mediator Snail, c-Myc downregulates TGF-β1 and TGF-β receptor 2, and Klf4 induces epithelial genes including E-cadherin. Blocking MET impairs the reprogramming of fibroblasts whereas preventing EMT in epithelial cells cultured with serum can produce iPSCs without Klf4 and c-Myc. Our work not only establishes MET as a key cellular mechanism toward induced pluripotency, but also demonstrates iPSC generation as a cooperative process between the defined factors and the extracellular milieu.
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► Mouse fibroblast reprogramming involves a mesenchymal-to-epithelial transition ► Sox2, Oct4, and c-Myc suppress TGF-β signaling ► Klf4 activates multiple epithelial genes including E-cadherin ► E-cadherin knockdown impairs reprogramming
MCPH1 is a causal gene for the neurodevelopmental disorder, human primary microcephaly (MCPH1, OMIM251200). Most pathogenic mutations are located in the N-terminal region of the gene, which encodes a ...BRCT domain, suggesting an important function of this domain in brain size determination. To investigate the specific function of the N-terminal BRCT domain in vivo, we generated a mouse model lacking the N'-BRCT domain of MCPH1 (referred as Mcph1-ΔBR1). These mutant mice are viable, but exhibit reduced brain size, with a thinner cortex due to a reduction of neuroprogenitor populations and premature neurogenic differentiation. Mcph1-ΔBR1 mice (both male and female) are infertile; however, almost all female mutants develop ovary tumours. Mcph1-ΔBR1 MEF cells exhibit a defect in DNA damage response and DNA repair, and show the premature chromosome condensation (PCC) phenotype, a hallmark of MCPH1 patient cells and also Mcph1 knockout cells. In comparison with Mcph1 complete knockout mice, Mcph1-ΔBR1 mice faithfully reproduce all phenotypes, indicating an essential role of the N-terminal BRCT domain for the physiological function of MCPH1 in the control of brain size and gonad development as well as in multiple cellular processes.
The p.G2019S mutation of the leucine-rich repeat kinase 2 (LRRK2) has been identified as the most prevalent genetic cause of familial and sporadic Parkinson's disease (PD). The Cre-LoxP recombination ...system has been used to correct the LRRK2-G2019S mutation in patient derived human induced pluripotent stem cells (hiPSCs) in order to generate isogenic controls. However, the remaining LoxP site can influence gene expression. In this study, we report the generation of a footprint-free LRRK2-G2019S isogenic hiPS cell line edited with the CRISPR/Cas9 and piggyBac technologies. We observed that the percentage of Tyrosine Hydroxylase (TH) positive neurons with a total neurite length of >2000μm was significantly reduced in LRRK2-G2019S dopaminergic (DA) neurons. The average branch number in LRRK2-G2019S DA neurons was also decreased. In addition, we have shown that in vitro TH positive neurons with a total neurite length of >2000μm were positive for Serine 129 phosphorylated (S129P) alpha-Synuclein (αS) and we hypothesize that S129P-αS plays a role in the maintenance or formation of long neurites. In summary, our footprint-free LRRK2-G2019S isogenic cell lines allow standardized, genetic background independent, in vitro PD modeling and provide new insights into the role of LRRK2-G2019S and S129P-αS in the pathogenesis of PD.
•Generation the first footprint-free LRRK2-G2019S isogenic cell line•LRRK2-G2019S reduces dopaminergic neurite complexity.•Detection of phosphorylated alpha-Synuclein in neurons with particularly long neurites•Reduction in the amount of S129P-a-Syn neurites upon presence of LRRK2-G2019S•Rescue of Parkinson's disease associated cellular phenotypes by drug treatment.
Genome editing and human induced pluripotent stem cells hold great promise for the development of isogenic disease models and the correction of disease-associated mutations for isogenic tissue ...therapy. CRISPR-Cas9 has emerged as a versatile and simple tool for engineering human cells for such purposes. However, the current protocols to derive genome-edited lines require the screening of a great number of clones to obtain one free of random integration or on-locus non-homologous end joining (NHEJ)-containing alleles. Here, we describe an efficient method to derive biallelic genome-edited populations by the use of fluorescent markers. We call this technique FACS-assisted CRISPR-Cas9 editing (FACE). FACE allows the derivation of correctly edited polyclones carrying a positive selection fluorescent module and the exclusion of non-edited, random integrations and on-target allele NHEJ-containing cells. We derived a set of isogenic lines containing Parkinson's-disease-associated mutations in α-synuclein and present their comparative phenotypes.
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•Fluorescent protein-SNP pairs enable deterministic genotypes for genome editing•Repetitive elements modulate off-targeting random integration•Parkinson's disease NESCs present impaired mitochondrial energy performance
In this article, Arias-Fuenzalida and colleagues show a platform to achieve deterministic genotypes for genome editing. The combinatorial use of fluorescent proteins and defined SNPs facilitates the genome editing endeavor. Using neuroepithelial stem cells, they demonstrate early mitochondrial phenotypes in SNCA mutants.
Parkinson's disease (PD)‐specific neurons, grown in standard 2D cultures, typically only display weak endophenotypes. The cultivation of PD patient‐specific neurons, derived from induced pluripotent ...stem cells carrying the LRRK2‐G2019S mutation, is optimized in 3D microfluidics. The automated image analysis algorithms are implemented to enable pharmacophenomics in disease‐relevant conditions. In contrast to 2D cultures, this 3D approach reveals robust endophenotypes. High‐content imaging data show decreased dopaminergic differentiation and branching complexity, altered mitochondrial morphology, and increased cell death in LRRK2‐G2019S neurons compared to isogenic lines without using stressor agents. Treatment with the LRRK2 inhibitor 2 (Inh2) rescues LRRK2‐G2019S‐dependent dopaminergic phenotypes. Strikingly, a holistic analysis of all studied features shows that the genetic background of the PD patients, and not the LRRK2‐G2019S mutation, constitutes the strongest contribution to the phenotypes. These data support the use of advanced in vitro models for future patient stratification and personalized drug development.
The identification of robust phenotypes recapitulating key cellular defects of Parkinson's disease (PD) is hampered by the lack of sufficiently representative in vitro models. Here, the cultivation of PD patients' derived dopaminergic neurons carrying the LRRK2‐G2019S mutation in 3D microfluidics is optimized. This system shows time‐dependent differentiation, viability, and mitochondrial phenotypes in LRRK2‐G2019S compared to LRRK2‐WT.
Emerging evidence suggests that Parkinson's disease (PD), besides being an age-associated disorder, might also have a neurodevelopment component. Disruption of mitochondrial homeostasis has been ...highlighted as a crucial cofactor in its etiology. Here, we show that PD patient-specific human neuroepithelial stem cells (NESCs), carrying the LRRK2-G2019S mutation, recapitulate key mitochondrial defects previously described only in differentiated dopaminergic neurons. By combining high-content imaging approaches, 3D image analysis, and functional mitochondrial readouts we show that LRRK2-G2019S mutation causes aberrations in mitochondrial morphology and functionality compared with isogenic controls. LRRK2-G2019S NESCs display an increased number of mitochondria compared with isogenic control lines. However, these mitochondria are more fragmented and exhibit decreased membrane potential. Functional alterations in LRRK2-G2019S cultures are also accompanied by a reduced mitophagic clearance via lysosomes. These findings support the hypothesis that preceding mitochondrial developmental defects contribute to the manifestation of the PD pathology later in life.
•Mitochondrial gene expression is altered in NESCs carrying the LRRK2-G2019 mutation•LRRK2-G2019S mutation induces alterations in mitochondrial morphology in NESCs•Mitophagy is affected in PD-specific NESCs carrying the LRRK2-G2019S mutation•Mitochondrial phenotypes in NESC are rescued by genetic correction of LRRK2-G2019S
Walter, Bolognin and colleagues show the detection of mitochondrial phenotypes in NESCs derived from Parkinson's disease (PD) patients carrying the LRRK2-G2019S mutation. This supports the use of stem cells as a relevant model to study PD-associated mitochondrial defects associated to PD.
The development of new and easy-to-use nucleases, such as CRISPR/Cas9, made tools for gene editing widely accessible to the scientific community. Cas9-based gene editing protocols are robust for ...creating knock-out models, but the generation of single nucleotide transitions or transversions remains challenging. This is mainly due to the low frequency of homology directed repair, which leads to the screening of a high number of clones to identify positive events. Moreover, lack of simultaneous biallelic modifications, frequently results in second-allele indels. For example, while one allele might undergo homology directed repair, the second can undergo non-homologous end joining repair. Here we present a step-wise protocol for biallelic gene editing. It uses two donors carrying a combination of fluorescent reporters alongside homology arms directed to the same genomic region for biallelic targeting. These homology arms carry the desired composite of modifications to be introduced (homozygous or heterozygous changes). Plus, the backbone of the plasmid carries a third fluorescent reporter for negative selection (to discard random integration events). Fluorescent selection of non-random biallelic targeted clones can be performed by microscopy guided picking or cell sorting (FACS). The positive selection module (PSM), carrying the fluorescence reporter and an antibiotic resistance, is flanked by inverted terminal repeats (ITR) that are recognized by transposase. Upon purification of the clones correctly modified, transfection of the excision-only transposase allows the removal of the PSM resulting in the integration of only the desired modifications.
Increasing evidence suggests that neurodevelopmental alterations might contribute to increase the susceptibility to develop neurodegenerative diseases. We investigate the occurrence of developmental ...abnormalities in dopaminergic neurons in a model of Parkinson’s disease (PD). We monitor the differentiation of human patient-specific neuroepithelial stem cells (NESCs) into dopaminergic neurons. Using high-throughput image analyses and single-cell RNA sequencing, we observe that the PD-associated LRRK2-G2019S mutation alters the initial phase of neuronal differentiation by accelerating cell-cycle exit with a concomitant increase in cell death. We identify the NESC-specific core regulatory circuit and a molecular mechanism underlying the observed phenotypes. The expression of NR2F1, a key transcription factor involved in neurogenesis, decreases in LRRK2-G2019S NESCs, neurons, and midbrain organoids compared to controls. We also observe accelerated dopaminergic differentiation in vivo in NR2F1-deficient mouse embryos. This suggests a pathogenic mechanism involving the LRRK2-G2019S mutation, where the dynamics of dopaminergic differentiation are modified via NR2F1.
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•LRRK2-G2019S accelerates cell-cycle exit and dopaminergic differentiation•LRRK2-G2019S reduces the viability of differentiating dopaminergic neurons•LRRK2-G2019S downregulates the core regulatory circuit transcription factor NR2F1•Dopaminergic differentiation is accelerated in NR2F1 mutant mouse embryos
Walter et al. show that differentiating iPSC-derived neurons carrying the LRRK2-G2019 mutation are characterized by faster initial neuronal differentiation and cell-cycle exit, as well as increased cell death, compared to controls. Downregulation of the transcription factor NR2F1 appears key to mediate LRRK2-G2019S-dependent phenotypes.
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