generation-of-human-brain-organoids-for-mitochondrial-disease-modeling

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...

generation-downstream-analysis-single-cell-single-nuclei

Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene ...

Max Delbrueck Center for Molecular Medicine (MDC)

Max Delbrück Center for Molecular Medicine (MDC)

In recent years freshwater flatworms (planarian) have become a powerful model for studies of regeneration and stem cell biology. Whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH) are key and most commonly used techniques to determine and visualize gene expression patterns in planaria. Here, we present the established version of whole-mount in situ hybridization (WISH) and whole-mount fluorescence in situ hybridization (WFISH) protocol optimized over the last years by several labs from the rapidly growing planaria field and give an overview of recently introduced modifications which can be critical in the study of low abundant transcripts. 

Whole-mount in situ hybridization using DIG-labeled probes in planarian.

The endoribonuclease Dicer is known for its central role in the biogenesis of eukaryotic small RNAs/microRNAs. Despite its importance, Dicer target transcripts have not been directly mapped. Here, we apply biochemical methods to human cells and C. elegans and identify thousands of Dicer-binding sites. We find known and hundreds of additional miRNAs with high sensitivity and specificity. We also report structural RNAs, promoter RNAs, and mitochondrial transcripts as Dicer targets. Interestingly, most Dicer-binding sites reside on mRNAs/lncRNAs and are not significantly processed into small RNAs. These passive sites typically harbor small, Dicer-bound hairpins within intact transcripts and generally stabilize target expression. We show that passive sites can sequester Dicer and reduce microRNA expression. mRNAs with passive sites were in human and worm significantly associated with processing-body/granule function. Together, we provide the first transcriptome-wide map of Dicer targets and suggest conserved binding modes and functions outside of the miRNA pathway.

A variety of dicer substrates in human and C. elegans.

Circular RNAs (circRNAs) are an endogenous class of animal RNAs. Despite their abundance, their function and expression in the nervous system are unknown. Therefore, we sequenced RNA from different brain regions, primary neurons, isolated synapses, as well as during neuronal differentiation. Using these and other available data, we discovered and analyzed thousands of neuronal human and mouse circRNAs. circRNAs were extraordinarily enriched in the mammalian brain, well conserved in sequence, often expressed as circRNAs in both human and mouse, and sometimes even detected in Drosophila brains. circRNAs were overall upregulated during neuronal differentiation, highly enriched in synapses, and often differentially expressed compared to their mRNA isoforms. circRNA expression correlated negatively with expression of the RNA-editing enzyme ADAR1. Knockdown of ADAR1 induced elevated circRNA expression. Together, we provide a circRNA brain expression atlas and evidence for important circRNA functions and values as biomarkers.

Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed.

Hundreds of circular RNAs (circRNAs) are highly abundant in the mammalian brain, often with conserved expression. Here we show that the circRNA Cdr1as is massively bound by the microRNAs (miRNAs) miR-7 and miR-671 in human and mouse brains. When the  locus was removed from the mouse genome, knockout animals displayed impaired sensorimotor gating-a deficit in the ability to filter out unnecessary information-which is associated with neuropsychiatric disorders. Electrophysiological recordings revealed dysfunctional synaptic transmission. Expression of miR-7 and miR-671 was specifically and posttranscriptionally misregulated in all brain regions analyzed. Expression of immediate early genes such as , a direct miR-7 target, was enhanced in -deficient brains, providing a possible molecular link to the behavioral phenotype. Our data indicate an in vivo loss-of-function circRNA phenotype and suggest that interactions between Cdr1as and miRNAs are important for normal brain function.

Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function.

Individual mRNA molecules can be imaged in fixed cells by hybridization with multiple, singly labeled oligonucleotide probes, followed by computational identification of fluorescent signals. This approach, called single-molecule RNA fluorescence in situ hybridization (smRNA FISH), allows subcellular localization and absolute quantification of RNA molecules in individual cells. Here, we describe a simple smRNA FISH protocol for two-color imaging of a circular RNA, CDR1as, simultaneously with an unrelated messenger RNA. The protocol can be adapted to circRNAs that coexist with overlapping, noncircular mRNA isoforms produced from the same genetic locus.

Single-Molecule Fluorescence In Situ Hybridization (FISH) of Circular RNA CDR1as.

Leigh syndrome (LS) is a severe manifestation of mitochondrial disease in children and is currently incurable. The lack of effective models hampers our understanding of the mechanisms underlying the neuronal pathology of LS. Using patient-derived induced pluripotent stem cells and CRISPR/Cas9 engineering, we developed a human model of LS caused by mutations in the complex IV assembly gene SURF1. Single-cell RNA-sequencing and multi-omics analysis revealed compromised neuronal morphogenesis in mutant neural cultures and brain organoids. The defects emerged at the level of neural progenitor cells (NPCs), which retained a glycolytic proliferative state that failed to instruct neuronal morphogenesis. LS NPCs carrying mutations in the complex I gene NDUFS4 recapitulated morphogenesis defects. SURF1 gene augmentation and PGC1A induction via bezafibrate treatment supported the metabolic programming of LS NPCs, leading to restored neuronal morphogenesis. Our findings provide mechanistic insights and suggest potential interventional strategies for a rare mitochondrial disease.

Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder that heavily burdens healthcare systems worldwide. There is a significant requirement to understand the still unknown molecular mechanisms underlying AD. Current evidence shows that two of the major features of AD are transcriptome dysregulation and altered function of RNA binding proteins (RBPs), both of which lead to changes in the expression of different RNA species, including microRNAs (miRNAs), circular RNAs (circRNAs), long non-coding RNAs (lncRNAs), and messenger RNAs (mRNAs). In this review, we will conduct a comprehensive overview of how RNA dynamics are altered in AD and how this leads to the differential expression of both short and long RNA species. We will describe how RBP expression and function are altered in AD and how this impacts the expression of different RNA species. Furthermore, we will also show how changes in the abundance of specific RNA species are linked to the pathology of AD.

RNA Dynamics in Alzheimer's Disease.

In the past decade, single-cell technologies have proliferated and improved from their technically challenging beginnings to become common laboratory methods capable of determining the expression of thousands of genes in thousands of cells simultaneously. The field has progressed by taking the CNS as a primary research subject - the cellular complexity and multiplicity of neuronal cell types provide fertile ground for the increasing power of single-cell methods. Current single-cell RNA sequencing methods can quantify gene expression with sufficient accuracy to finely resolve even subtle differences between cell types and states, thus providing a great tool for studying the molecular and cellular repertoire of the CNS and its disorders. However, single-cell RNA sequencing requires the dissociation of tissue samples, which means that the interrelationships between cells are lost. Spatial transcriptomic methods bypass tissue dissociation and retain this spatial information, thereby allowing gene expression to be assessed across thousands of cells within the context of tissue structural organization. Here, we discuss how single-cell and spatially resolved transcriptomics have been contributing to unravelling the pathomechanisms underlying brain disorders. We focus on three areas where we feel these new technologies have provided particularly useful insights: selective neuronal vulnerability, neuroimmune dysfunction and cell-type-specific treatment response. We also discuss the limitations and future directions of single-cell and spatial RNA sequencing technologies.

Single-cell and spatial transcriptomics: deciphering brain complexity in health and disease.

Herpes simplex encephalitis is a life-threatening disease of the central nervous system caused by herpes simplex viruses (HSVs). Following standard of care with antiviral acyclovir treatment, most patients still experience various neurological sequelae. Here we characterize HSV-1 infection of human brain organoids by combining single-cell RNA sequencing, electrophysiology and immunostaining. We observed strong perturbations of tissue integrity, neuronal function and cellular transcriptomes. Under acyclovir treatment viral replication was stopped, but did not prevent HSV-1-driven defects such as damage of neuronal processes and neuroepithelium. Unbiased analysis of pathways deregulated upon infection revealed tumour necrosis factor activation as a potential causal factor. Combination of anti-inflammatory drugs such as necrostatin-1 or bardoxolone methyl with antiviral treatment prevented the damages caused by infection, indicating that tuning the inflammatory response in acute infection may improve current therapeutic strategies.

Modelling viral encephalitis caused by herpes simplex virus 1 infection in cerebral organoids.

Alzheimer's disease (AD) is characterized by the intra- and extracellular accumulation of amyloid-β (Aβ) peptides. How Aβ aggregates perturb the proteome in brains of patients and AD transgenic mouse models, remains largely unclear. State-of-the-art mass spectrometry (MS) methods can comprehensively detect proteomic alterations, providing relevant insights unobtainable with transcriptomics investigations. Analyses of the relationship between progressive Aβ aggregation and protein abundance changes in brains of 5xFAD transgenic mice have not been reported previously.

A proteomics analysis of 5xFAD mouse brain regions reveals the lysosome-associated protein Arl8b as a candidate biomarker for Alzheimer's disease.

Organoids derived from stem cells have become an increasingly important tool for studying human development and modeling disease. However, methods are still needed to control and study spatiotemporal patterns of gene expression in organoids. Here we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes in programmable spatiotemporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. Spatial and single-cell transcriptomic analyses showed that this local induction was sufficient to generate stereotypically patterned organoids and revealed new insights into SHH's contribution to gene regulation in neurodevelopment. With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.

ArticleTotal : 2	Year
Publication title Cited by 9	2021
Publication title	2024

Article	Year
Whole-mount in situ hybridization using DIG-labeled probes in planarian. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 25218375	2014
A variety of dicer substrates in human and C. elegans. Cell\| PubMed ID: 25416952	2014
Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed. Molecular cell\| PubMed ID: 25921068	2015
Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function. Science (New York, N.Y.)\| PubMed ID: 28798046	2017
Single-Molecule Fluorescence In Situ Hybridization (FISH) of Circular RNA CDR1as. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29322442	2018
Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome. Nature communications\| PubMed ID: 33771987	2021
RNA Dynamics in Alzheimer's Disease. Molecules (Basel, Switzerland)\| PubMed ID: 34500547	2021
Single-cell and spatial transcriptomics: deciphering brain complexity in health and disease. Nature reviews. Neurology\| PubMed ID: 37198436	2023
Modelling viral encephalitis caused by herpes simplex virus 1 infection in cerebral organoids. Nature microbiology\| PubMed ID: 37349587	2023
A proteomics analysis of 5xFAD mouse brain regions reveals the lysosome-associated protein Arl8b as a candidate biomarker for Alzheimer's disease. Genome medicine\| PubMed ID: 37468900	2023
Spatiotemporal, optogenetic control of gene expression in organoids. Nature methods\| PubMed ID: 37735569	2023

Agnieszka Rybak-Wolf

Biography

JoVE Journal Publications

Other Publications