Projection neuron vulnerability suggests a therapeutic path


Region- and lineage-specific transcriptomic changes appear to be responsible for the characteristic pathobiology of multiple sclerosis (MS).

A study that used single-nucleus RNA sequencing to assess changes in cell lineages in MS lesions has shed light on the selective cortical neuron damage and glial activation that contribute to the progression of MS lesions.

In this research, Lucas Schirmer, MD, David Rowitch, MD, PhD, and colleagues at the University of Cambridge (England) and University of California, San Francisco, found selective vulnerability and loss of projection neurons in the upper-cortical layers of MS patients, with associated inflammation mediated by B cells. The team’s findings support further investigation of anti–B-cell biological therapies for MS. Beyond the findings themselves, the techniques used in the study mark an advancement in the spatial resolution of neuropathologic processes.

Future work is needed to determine whether this biology is beneficial or detrimental in disease course, for example, by exacerbating inflammation.

“This study builds on progress from projects such as the human cell atlas and BRAIN initiative [that] have helped develop single-cell transcriptomic technologies,” said Dr. Rowitch, principal investigator from the University of Cambridge. “Here, we have applied these technologies to the normal human brain, but importantly, also to the human brain affected by [MS]. We report a new technique, large area spatial transcriptomic mapping, that shows gene expression at single cell resolution but over large areas that capture the heterogeneity of human brain lesions in MS.”

Although it is widely known that cortical gray matter is demyelinated and lost in MS, particularly beneath inflamed meninges, it is poorly understood if all cortical neurons are affected, or just certain neuronal subtypes. “Indeed, cell type–specific mechanisms of MS progression, including scar formation with slowly expanding [white matter] lesions and cortical atrophy are not well understood,” the investigators wrote (Nature. 2019 Jul 17. doi: 10.1038/s41586-019-1404-z).

Their study involved comparison of frozen brain tissue samples from 17 patients with MS and 16 healthy controls. The samples came from MS patients who did not receive modern immunomodulatory therapies, thus they represented the endpoint of natural disease course. Final analysis involved 12 MS samples and 9 control samples, including sections of cortical gray matter and subcortical white matter, plus meningeal tissue. RNA gene expression was assessed with single-nucleus RNA sequencing and validated with fluorescence multiplex in situ hybridization. Multiple brain cell types (neurons, oligodendrocytes, microglia, and astrocytes) were analyzed across diverse lesional and nonlesional areas.

Credit: Juan Gaertner/Science Source
Microglia cells scavenging the white matter of the brain for infectious agents, plaques or damaged neurons. They are involved in autoimmune diseases like multiple sclerosis and Alzheimer’s disease.

The RNA sequencing found nearly 49,000 single-nucleus profiles and 22 cell clusters, none of which comprised nuclei from a single MS or control sample. The samples from MS patients with cortical demyelination had a selective reduction in excitatory neurons in the upper layer. In contrast, excitatory neurons in the intermediate and deep layers were similar in number for MS and control samples.

Neurons from all cortical layers showed enrichment of cell stress pathways in MS patients, compared with controls.

Selective vulnerability and loss of CUX2-expressing L2-L3 excitatory neurons were noted in the upper-layer neurons of patients with MS, a phenomenon that correlated with known patterns of degeneration. High B-cell activity was found in involved lesions, spotlighting the role of B cells in the destruction of projection neurons.

Theoretically, anti–B-cell biological therapies could protect projection neurons and slow disease progression, Dr. Rowitch said.

He also noted that the relevance of these findings may extend beyond MS. “In an earlier study of brain cells from people with autism spectrum disorder, we also found changes in the same projection neurons, even though MS and autism affect the brain very differently,” Dr. Rowitch said. “Why would the same neuron be popping up in these two very different situations? We’re really interested in exploring that further.”

Distinct transcripts were also detected in cortical astrocytes as compared to subcortical lesion astrocytes, suggesting different tissue microenvironments at a molecular level. RNA sequencing was also able to home in on phagocytosing cells in MS based on transport of ingested myelin transcripts to the nucleus and perinuclear structures.

“Future work is needed to determine whether this biology is beneficial or detrimental in disease course, for example, by exacerbating inflammation,” the investigators wrote.

In addition to expanding the understanding of MS disease processes and pointing to new research topics, the techniques involved in the study mark a new level of precision in MS research. “This work opens up MS research to direct exploration of cell damage pathways based on high resolution single cell data,” Dr. Rowitch said. “With regard to the neurons that we find are lost, we believe we can trace the cell death pathways shown by our data to develop new cell type–specific neuroprotective therapies to work in conjunction with conventional therapies for MS.”

The study was funded by the National Multiple Sclerosis Society, the National Institutes of Health, Wellcome Trust and others. The investigators reported no conflicts of interest.