Researchers Use Single Nucleus and Single Cell Techniques to Understand Neurological Disorders
The central nervous system, with its complex network of distinct cell types and connections, poses significant health challenges. Complex neurological disorders that exhibit clinical and genetic heterogeneity, such as autism and multiple sclerosis are often much more difficult to diagnose than other diseases, presenting symptoms that can vary greatly among patients. Further, the biological mechanisms underlying these disorders are poorly understood and difficult to study, defying concentrated attempts to determine their causes and develop treatments.
However, single cell gene expression profiling, and its close cousin single nucleus gene expression, are now helping scientists make significant progress in understanding the causes and pathogenesis of these disorders and may someday lead to effective treatments.
Researchers, including David Rowitch, neuroscientist and professor of pediatrics at the University of California, San Francisco (UCSF), and Arnold Kriegstein, neuroscientist and director of the UCSF Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, have made recent progress in studying neurological disorders. The team used 10x Genomics single cell techniques to identify molecular changes in autism spectrum disorder and multiple sclerosis that seem to exist only in specific cell types. Traditional genomics studies require hundreds of cells as samples, forcing researchers to take average readings of cellular activity across cell populations, even among diverse groups of cells, and cannot identify rare cell types or changes in individual cells.
Single cell gene expression is often not compatible with this type of neuroscience research, which relies on frozen brain and neural tissue samples that have reduced cell quality and increased RNA damage. Fortunately, single nuclei, isolated from frozen and fixed tissue samples, can be used with the 10x Genomics Chromium and Cell Ranger systems, bypassing the degradation and quality problems associated with frozen samples.
Single cell (or nucleus) genomics makes it possible to examine differences in gene expression between distinct neural cell types, such as excitatory and inhibitory neurons, astrocytes, oligodendrocytes, and other glial cells. Each of these cell types play vital roles in the working of a healthy nervous system, exhibit distinct behaviors, and, as UCSF researchers have shown, act differently in a diseased state.
In May of this year, the team published a study in Science that found changes in autism spectrum disorder that were tied to specific cells. Using snRNA-seq and the 10x Genomics platform, they were able to, for the first time, determine the specific cell types in the brain affected by autism spectrum disorder (1). The team found 692 events in 513 unique differentially expressed genes. Of those, 407 were expressed in a single cell type. Key genes were overrepresented in L2/3 and L4 (all upper levels of the cortex) excitatory neurons, as well as VIP- and somatostatin-expressing interneurons, which set daily cellular rhythms and regulate endocrine functions, respectively. These gene dysregulations in upper-layer cortical neurons and microglia were correlated with the clinical severity of autism cases, demonstrating that such molecular changes in upper cortical networks are closely linked to the disorder.
Just two months after their autism study was published, the team published another article in Nature describing a single cell study of multiple sclerosis (MS). They found that neurons are differentially vulnerable to damage from MS lesions and that the most dysregulated genes in the disease appear in upper cortical neurons and certain reactive glial cells located on the edge of MS lesions in the subcortex (2). Single nucleus RNA-sequencing (snRNA-seq) identified more than 48000 single nuclear profiles, and about 1400 genes and 2400 transcription products per nucleus. The 10x Genomics Chromium Single Cell Gene Expression Solution distinguished phagocytosing cells in multiple sclerosis by their transportation of ingested myelin transcripts (products of gene expression) into the cell nucleus. From these profiles, they found that snRNA-seq was a valuable method for determining the extent of heterogeneous MS lesions and specific demyelination and inflammation.
These recent studies demonstrate the power of single cell and single nucleus gene expression profiling in studying complex neurological disorders. We can’t wait to see how researchers use it next.
- D. Velmeshev, L. Schirmer, D. Jung et al., Single-cell genomics identifies cell type-specific molecular changes in autism. Science. 364, 685–689 (2019).
- L. Schirmer, D. Velmeshev, S. Holmqvist et al., Neuronal vulnerability and multilineage diversity in multiple sclerosis. Nature. 573 (2019).