Researchers have integrated two sequencing technologies in individual cells to discover new variations in mRNAs linked to Alzheimer’s disease, dementia with Lewy bodies, and Parkinson’s disease.
Neurodegenerative diseases, which lead to the gradual decline of brain function, present significant challenges in understanding and treatment. These prevalent conditions impact millions worldwide; however, attempts to create new therapies have largely fallen short.
At Sanford Burnham Prebys, scientists are exploring new avenues for potential treatments by gaining insights into how neurodegenerative diseases affect brain cells.
Led by Jerold Chun, MD, PhD, a professor in the Degenerative Diseases Program at Sanford Burnham Prebys, the research results were published on December 10, 2024, in eNeuro. The study involved combining two single-cell sequencing technologies to identify new distinctions in mRNAs associated with Alzheimer’s disease (AD), dementia with Lewy bodies (DLB), and Parkinson’s disease (PD). Genes can generate multiple types of messenger RNA (mRNA) through a process called alternative splicing, leading to several protein forms, which are referred to as isoforms.
The research team implemented two forms of single-nucleus RNA sequencing (snRNAseq) in this study, building on a significant 2016 publication in Science by Chun and his collaborators regarding the use of snRNAseq in human brain research.
“Currently, snRNAseq is the benchmark for analyzing single-cell transcriptomes in human brains,” Chun stated. “Given the brain’s intricate mix of cells with countless connections, other single-cell technologies are more prone to contamination from surrounding materials.”
This challenge is addressed by snRNAseq, which isolates the nuclei of each cell in a sample, allowing researchers to examine the RNA molecules that contain the genetic blueprints for creating new proteins.
“Nevertheless, standard single-cell sequencing typically utilizes short-read sequencing,” explained Christine Liu, PhD, a postdoctoral researcher in Chun’s lab and the study’s primary author. “This approach reads 100 to 150 base pairs at a time, comparing them to a reference genome.”
These comparisons help map the shorter sequences to a reference sequence. Any variations from the reference genome are termed variants. However, using short reads to reconstruct the overall picture has its limitations.
“Short-read sequencing struggles with specific types of sequence variants. To improve our capture of these, we also employed long-read sequencing, which reads from 5,000 to 30,000 base pairs at once and does not depend on mapping to a reference genome,” said Chun.
The research team applied both methods to single cells from post-mortem brain tissue of 25 donors diagnosed with either AD, DLB, or PD, as well as samples from healthy donor brains serving as a control. An analysis of over 165,000 cells was conducted, focusing on long-read sequencing of mRNAs from the 50 genes most associated with these neurodegenerative diseases in previous studies.
The results revealed new mRNA sequences from all 50 targeted genes, which had not been previously identified through earlier sequencing efforts.
“By integrating both short- and long-read sequencing, we observed significant mRNA isoform diversity in these genes, including those that did not show differential expression in short-read data,” noted Liu. “In some instances, the newly identified transcripts appear to constitute a large portion of the total isoforms.”
“Our findings reinforce earlier discoveries that around 75% of mRNAs in the brain transcriptome remain unknown,” Chun remarked, referring to a 2021 PNAS paper that highlighted the discovery of hundreds of thousands of new mRNA transcripts. “There is still much to learn about these new mRNAs and how they evolve with disease.”
The research team is also investigating the types of novel proteins produced from these transcripts.
“The emergence of new mRNA isoforms suggests the production of new proteins in diseased brains and cells,” said Chun, “which may uncover previously unrecognized targets for therapeutic intervention in efforts to find treatments for these widespread and debilitating diseases.”
Additional contributors to the study from Sanford Burnham Prebys include Chris Park, Tony Ngo, Janani Saikumar, Carter R. Palmer, Anis Shahnaee, and William J. Romanow.
This research was funded by the National Institutes of Health (R01AG071465 and R01AG065541), the National Institute of General Medical Sciences (T32GM007752), the U.S. Department of Defense (W81XWH-21-10642), and several foundations including Bruce Ford and Anne Smith Bundy Foundation, Larry L. Hillblom Foundation, and the California Institute for Regenerative Medicine (EDUC4-12813-01).