Single-cell DNA methylation analysis tool Amethyst reveals distinct noncanonical methylation patterns in human glial cells.

Single-cell sequencing technologies have revolutionized biomedical research by enabling deconvolution of cell type-specific properties in highly heterogeneous tissue. While robust tools have been developed to handle bioinformatic challenges posed by single-cell RNA and ATAC data, options for emergent modalities such as methylation are much more limited, impeding the utility of results. Here we present Amethyst, a comprehensive R package for atlas-scale single-cell methylation sequencing data analysis. Amethyst begins with base-level methylation calls and expedites batch integration, doublet detection, dimensionality reduction, clustering, cell type annotation, differentially methylated region calling, and interpretation of results, facilitating rapid data interaction in a local environment. We introduce the workflow using published single-cell methylation human peripheral blood mononuclear cell (PBMC) and human cortex data. We further leverage Amethyst on an atlas-scale brain dataset to discover a noncanonical methylation pattern in human astrocytes and oligodendrocytes, challenging prior assumptions that this form of methylation is only biologically relevant to neurons in the brain.

sciMET-cap: high-throughput single-cell methylation analysis with a reduced sequencing burden.

DNA methylation is a key component of the mammalian epigenome, playing a regulatory role in development, disease, and other processes. Robust, high-throughput single-cell DNA methylation assays are now possible (sciMET); however, the genome-wide nature of DNA methylation results in a high sequencing burden per cell. Here, we leverage target enrichment with sciMET to capture sufficient information per cell for cell type assignment using substantially fewer sequence reads (sciMET-cap). Accumulated off-target coverage enables genome-wide differentially methylated region (DMR) calling for clusters with as few as 115 cells. We characterize sciMET-cap on human PBMCs and brain (middle frontal gyrus).

iPSC-derived models of PACS1 syndrome reveal transcriptional and functional deficits in neuron activity.

PACS1 syndrome is a neurodevelopmental disorder characterized by intellectual disability and distinct craniofacial abnormalities resulting from a de novo p.R203W variant in phosphofurin acidic cluster sorting protein 1 (PACS1). PACS1 is known to have functions in the endosomal pathway and nucleus, but how the p.R203W variant affects developing neurons is not fully understood. Here we differentiated stem cells towards neuronal models including cortical organoids to investigate the impact of the PACS1 syndrome-causing variant on neurodevelopment. While few deleterious effects were detected in PACS1(+/R203W) neural precursors, mature PACS1(+/R203W) glutamatergic neurons exhibited impaired expression of genes involved in synaptic signaling processes. Subsequent characterization of neural activity using calcium imaging and multielectrode arrays revealed the p.R203W PACS1 variant leads to a prolonged neuronal network burst duration mediated by an increased interspike interval. These findings demonstrate the impact of the PACS1 p.R203W variant on developing human neural tissue and uncover putative electrophysiological underpinnings of disease.

PCDH12 loss results in premature neuronal differentiation and impeded migration in a cortical organoid model.

Protocadherins (PCDHs) are cell adhesion molecules that regulate many essential neurodevelopmental processes related to neuronal maturation, dendritic arbor formation, axon pathfinding, and synaptic plasticity. Biallelic loss-of-function variants in PCDH12 are associated with several neurodevelopmental disorders (NDDs). Despite the highly deleterious outcome resulting from loss of PCDH12, little is known about its role during brain development and disease. Here, we show that PCDH12 loss severely impairs cerebral organoid development, with reduced proliferative areas and disrupted laminar organization. 2D models further show that neural progenitor cells lacking PCDH12 prematurely exit the cell cycle and differentiate earlier when compared with wild type. Furthermore, we show that PCDH12 regulates neuronal migration and suggest that this could be through a mechanism requiring ADAM10-mediated ectodomain shedding and/or membrane recruitment of cytoskeleton regulators. Our results demonstrate a critical involvement of PCDH12 in cortical organoid development, suggesting a potential cause for the pathogenic mechanisms underlying PCDH12-related NDDs.

sciMET-cap: High-throughput single-cell methylation analysis with a reduced sequencing burden.

DNA methylation is a key component of the mammalian epigenome, playing a regulatory role in development, disease, and other processes. Robust, high-throughput single-cell DNA methylation assays are now possible (sciMET); however, the genome-wide nature of DNA methylation results in a high sequencing burden per cell. Here, we leverage target enrichment with sciMET to capture sufficient information per cell for cell type assignment using substantially fewer sequence reads (sciMET-cap). Sufficient off-target coverage further enables the production of near-complete methylomes for individual cell types. We characterize sciMET-cap on human PBMCs and brain (middle frontal gyrus).

Impaired migration and premature differentiation underlie the neurological phenotype associated with PCDH12 loss of function.

Protocadherins (PCDHs) are cell adhesion molecules that regulate many essential neurodevelopmental processes related to neuronal maturation, dendritic arbor formation, axon pathfinding, and synaptic plasticity. Bi-allelic loss-of-function variants in PCDH12 are associated with several neurodevelopmental disorders (NDDs) such as diencephalic-mesencephalic dysplasia syndrome, cerebral palsy, cerebellar ataxia, and microcephaly. Despite the highly deleterious outcome resulting from loss of PCDH12, little is known about its role during brain development and disease. Here, we show that PCDH12 loss severely impairs cerebral organoid development with reduced proliferative areas and disrupted laminar organization. 2D models further show that neural progenitor cells lacking PCDH12 prematurely exit cell cycle and differentiate earlier when compared to wildtype. Furthermore, we show that PCDH12 regulates neuronal migration through a mechanism requiring ADAM10-mediated ectodomain shedding and membrane recruitment of cytoskeleton regulators. Our data demonstrate a critical and broad involvement of PCDH12 in cortical development, revealing the pathogenic mechanisms underlying PCDH12-related NDDs.

In search of a cure: PACS1 Research Foundation as a model of rare disease therapy development.

Rare diseases affect nearly 400 million people worldwide and have a devastating impact on patients and families. Although these diseases are collectively common, they are often overlooked by the research community. We present the ongoing work of the PACS1 Syndrome Research Foundation as a paradigm for approaching rare disease research.

PYRC2-Related Hypomyelinating Leukodystrophy: More to This Than Meets the Eye.

Loss-of-function variants in the PYRC2 gene cause hypomyelinating leukodystrophy 10 (HLD10), but the associated pathogenic mechanisms are unknown. In this issue of Neuron, Escande-Beillard et al. (2020) reveal that PYRC2 is a key enzyme for proper brain development and a regulator of glycine homeostasis, uncovering hyperglycinemia as a driver of HLD10 pathogenesis.

Genetic Causes and Modifiers of Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is one of the most prevalent neurodevelopmental disorders, affecting an estimated 1 in 59 children. ASD is highly genetically heterogeneous and may be caused by both inheritable and de novo gene variations. In the past decade, hundreds of genes have been identified that contribute to the serious deficits in communication, social cognition, and behavior that patients often experience. However, these only account for 10-20% of ASD cases, and patients with similar pathogenic variants may be diagnosed on very different levels of the spectrum. In this review, we will describe the genetic landscape of ASD and discuss how genetic modifiers such as copy number variation, single nucleotide polymorphisms, and epigenetic alterations likely play a key role in modulating the phenotypic spectrum of ASD patients. We also consider how genetic modifiers can alter convergent signaling pathways and lead to impaired neural circuitry formation. Lastly, we review sex-linked modifiers and clinical implications. Further understanding of these mechanisms is crucial for both comprehending ASD and for developing novel therapies.

GLUT1 is associated with sphingolipid-organized, cholesterol-independent domains in L929 mouse fibroblast cells.

Glucose is a preferred metabolite in most mammalian cells, and proper regulation of uptake is critical for organism homeostasis. The glucose transporter 1 (GLUT1) is responsible for glucose uptake in a wide variety of cells and appears to be regulated in a tissue specific manner. Therefore, a better understanding of GLUT1 regulation within its various cellular environments is essential for developing therapeutic strategies to treat disorders associated with glucose homeostasis. Previous findings suggest that plasma membrane subdomains called lipid rafts may play a role in regulation of GLUT1 uptake activity. While studying this phenomenon in L929 mouse fibroblast cells, we observed that GLUT1 associates with a low density lipid microdomain distinct from traditionally-defined lipid rafts. These structures are not altered by cholesterol removal with methyl-ß-cyclodextrin and lack resistance to cold Triton X-100 extraction. Our data indicate that the GLUT1-containing membrane microdomains in L929 cells, as well as GLUT1's basal activity, are instead sphingolipid-dependent, being sensitive to both myriocin and sphingomyelinase treatment. These microdomains appear to be organized primarily by their lipid composition, as disruption of the actin cytoskeleton or microtubules does not alter the association of GLUT1 with them. Furthermore, the association of GLUT1 with these microdomains appears not to require palmitoylation or glycosylation, as pharmacologic inhibition of these processes had no impact on GLUT1 density in membrane fractions. Importantly, we find no evidence that GLUT1 is actively translocated into or out of low density membrane fractions in response to acute activation in L929 cell.

Mitochondria, ER, and nuclear membrane defects reveal early mechanisms for upper motor neuron vulnerability with respect to TDP-43 pathology.

Insoluble aggregates containing TDP-43 are widely observed in the diseased brain, and defined as "TDP-43 pathology" in a spectrum of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease and ALS with frontotemporal dementia. Here we report that Betz cells of patients with TDP-43 pathology display a distinct set of intracellular defects especially at the site of nuclear membrane, mitochondria and endoplasmic reticulum (ER). Numerous TDP-43 mouse models have been generated to discern the cellular and molecular basis of the disease, but mechanisms of neuronal vulnerability remain unknown. In an effort to define the underlying causes of corticospinal motor neuron (CSMN) degeneration, we generated and characterized a novel CSMN reporter line with TDP-43 pathology, the prp-TDP-43A315T-UeGFP mice. We find that TDP-43 pathology related intracellular problems emerge very early in the disease. The Betz cells in humans and CSMN in mice both have impaired mitochondria, and display nuclear membrane and ER defects with respect to TDP-43 pathology.