New system discovers brain defects in autism
Figure 1 – Schematic view of the CHOOSE system
Abstract
Neurodevelopmental disorders (NDDs), like autism spectrum disorder (ASD), are hard to study due to their arise in fetal stages. Furthermore, diagnoses are all after-birth, sometimes even after years. These diagnoses are all based on behavioral analysis, which are hard and often not precise. The need to develop new strategies to expand the knowledge on these diseases is imminent. The CRISPR-human organoids-single-cell RNA sequencing (CHOOSE) system tries to do exactly that with autism spectrum disorder. By applying CRISPR technology to recapitulate mutations in some ASD high-risk genes, the perturbed cells are used to produce brain organoids, which are then analyzed at single-cell resolution. This allows the simulation of the typical defects that arise in the neurodevelopment of ASD patients, helping to understand the gene regulatory network (GRN) involved in this disorder. The authors validated their findings in vivo on the brain of a patient through MRI.
Review
Neurodevelopmental disorders (NDDs) affect around 15% of children and adolescents worldwide, leading to a multitude of impairments, especially in cognition, communication, adaptive behavior and psychomotor skills. [1] The fifth edition of the Diagnostic and Statistical Manual of Mental Disorders categorize the following disorders as the main NDDs: intellectual disabilities (ID), communication disorder, autism spectrum disorder (ASD), attention deficit/hyperactivity disorder (ADHD), specific learning disorder and motor disorder. [1]
The molecular mechanisms behind the physiopathology of these NDDs are still mainly unknown, due to difficulties in a research field that is based mostly on after-death tissue analysis. This causes a slowdown in the knowledge on these diseases, leading to inefficient diagnostic techniques and a lack of potential therapies. In a recent study published in Nature in 2023, a new system is proposed to study the molecular physiopathology behind ASD, one of the most challenging NDDs, avoiding the use of post-mortem tissues or in vivo animal models.
Autism Spectrum Disorder
Over the last 40 years, the genetic knowledge of ASD has evolved enormously, where twin and family studies showed ASD to be one of the most highly heritable disorders. [2] These studies allowed to identify the most important genetic variants, and they have been collected in databases such as the Simons Foundation Autism Research Initiative (SFARI) one. Interestingly, around 2/3 of ASD-associated genetic variants of ASD are shared with those of ADHD. [3]
The Choose System
The CHOOSE system uses the CRISPR/Cas9 technology to induce the loss-of-function of 36 high-risk ASD genes found in the SFARI database. This is done through specific gRNAs, which are inserted in a human embryonic stem cell (hES) line expressing inducible eCas9 using lentiviruses. The endonuclease eCas9 is induced to perturb each specific gene, causing the activation of the repair mechanisms of the cell and leading to a frameshift mutation, thus blocking the expression of the gene.
After the generation of a pooled lentiviral library containing all the gRNAs targeting the high-risk genes, cells are transfected using a low infection rate (low MOI), to ensure that only one perturbation is induced per cell. The transfected cells are then used to generate telencephalic brain organoids, which offer an unprecedented opportunity to study the complex processes of human neurodevelopment.
After 120 days, organoids are dissociated and analyzed using multi-omics profiling at single-cell resolution thanks to the unique clone barcode (UCB) inserted with each gRNA, which allows to understand which gene is perturbed in each cell. This allows the study of the phenotypical differences in presence of the ASD-related genetic perturbations. [4]
Results
By comparing difference in gRNA abundances between induced and uninduced organoids across various batches (using both scRNA-seq complementary DNA and bulk DNA sequencing to recover gRNA information), the authors identified the gene perturbations that affect cell proliferation and survival.
They next analyzed the scRNA-seq data from CHOOSE organoids to investigate the effects of ASD gene perturbations on the transcriptome of each cell type, revealing the cell populations most vulnerable to these disruptions. They identified the layer 2/3 excitatory neurons (L2/3 ENs), interneuron precursor cells (INPs) and intermediate progenitor cells (IPCs) as the most vulnerable cell types.
Moreover, among all the analyzed perturbations, 2071 differentially expressed genes (DEGs) were detected by comparing each perturbation to control brain organoids from different batches. Interestingly, the TOP-DEGs were enriched in ASD-associated genes, as reported in the SFARI database, but not on the intellectual disability (ID) one (sysID database). This suggests a specificity in the molecular mechanisms of ASD.
Using single-cell multiome data and the R tool Pando, which integrates scRNA-seq and scATAC-seq data, gene regulatory modules were inferred and analyzed, allowing the reconstruction of the gene regulatory network (GRN) supporting telencephalic brain organoids development. This enabled the delineation of interactions between transcription factors and their target genes, including regulatory relationships among transcription factors themselves, and the assessment of the effects of ASD gene perturbations on such GRN.
Finally, the authors focused on a specific gene, ARID1B, which, when perturbed, caused a significant enrichment of ventral Radial Glial Cells (v-RGCs). Physiologically, this gene pushes v-RGCs to differentiate towards either interneurons or oligodendrocyte precursor cells (OPCs). The analysis of the differentiation trajectory showed that ARID1B perturbation causes a shift towards the oligodendrocyte’s direction, thus changing the differentiation fate of v-RGCSs. These results were further validated on brain organoids generated from iPS cell lines of 2 ASD patients carrying a heterozygous ARID1B mutation. Finally, by analyzing an in utero fetal MRI of one of the patients, an enlargement of the ganglionic eminence (GE) was shown, due to an increase in ventral progenitors. [4]
Conclusions
The application of this new system can be extended to research on all NDDs, allowing researchers to gain deeper knowledge into these still largely unknown disorders. But it doesn’t lack limitations that need to be revised and possibly fixed. The brain organoids, even if complex, can’t be compared with a real in vivo brain due to the lack of certain cell types, like the microglia or various interneurons (e.g. those forming the medial ganglionic eminence), which are absent in this model.
Also, the CRISPR technique is used to induce mutations, but it cannot specify if the perturbed cells are heterozygous or homozygous for the induced mutation. Some ASD-related genes can be heterozygous, while others are homozygous; therefore, a more precise analysis needs to be performed. Also, the organoids generated are mosaic, meaning that there are both perturbed and wild-type cells. This can generate competition between these different cells.
Overall, it is a good starting point for the development of a system that might be helpful in studying the developmental defects and how these manifest phenotypically. In the future, the use of this system could lead to a better characterization of cell-type specific defects during neurodevelopment, allowing applications – such as that presented with the MRI analysis of the ARID1B patient – which could one day also enable an intrauterine diagnosis, something that is still impossible for NDDs nowadays.
References
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- Rutter M. L. (2011). Progress in understanding autism: 2007-2010. Journal of autism and developmental disorders, 41(4), 395–404. https://doi.org/10.1007/s10803-011-1184-2
- Thapar, A., & Rutter, M. (2021). Genetic Advances in Autism. Journal of autism and developmental disorders, 51(12), 4321–4332. https://doi.org/10.1007/s10803-020- 04685-z
- Li, C., Fleck, J. S., Martins-Costa, C., Burkard, T. R., Themann, J., Stuempflen, M., Peer, A. M., Vertesy, Á., Littleboy, J. B., Esk, C., Elling, U., Kasprian, G., Corsini, N. S., Treutlein, B., & Knoblich, J. A. (2023). Single-cell brain organoid screening identifies developmental defects in autism. Nature, 621(7978), 373–380. https://doi.org/10.1038/s41586-023-06473-y