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Leng K, Li E, Eser R, Piergies A, Sit R, Tan M, Neff N, Li SH, Rodriguez RD, Suemoto CK, Leite RE, Ehrenberg AJ, Pasqualucci CA, Seeley WW, Spina S, Heinsen H, Grinberg LT, Kampmann M. Molecular characterization of selectively vulnerable neurons in Alzheimer's disease. Nat Neurosci. 2021 Feb;24(2):276-287. Epub 2021 Jan 11 PubMed.
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University of California, Los Angeles
In Alzheimer’s disease, which is so complex, selective cellular vulnerability is a key anchoring point for mechanistic discovery. To leverage it, we must understand what differs between vulnerable and spared cells as disease progresses.
Single-nuclear RNA sequencing of human disease tissue brings incredible new opportunities here—but specimen selection and validation are paramount. By using highly curated neuropathological specimens, and validation across independent studies, this important work sets an exciting foundation for continued discovery.
View all comments by Jessica RexachBanner Sun Health Research Institute
Banner Sun Health Research Institute
Translational Genomics Research Institute
This report is intriguing and reinforces the power of single-nucleus studies of human postmortem brain tissue for understanding neurodegenerative disease. The identification of RORB as a possibly critical protein associated with selective vulnerability may lead to new mechanistic insights into neurofibrillary tangle formation, if confirmed in other single-nucleus studies and hopefully as well by studies using other approaches to single-cell or defined-cell-type analysis.
The authors used CP13, marker of early to middle stages of neurofibrillary tangle development (Vingtdeux et al., 2011). Comparison with a late-stage tangle marker such as PHF-1 might be of assistance in sorting out the stage at which RORB becomes involved.
One of the major deficiencies of single-nucleus studies, however, is that there is the assumption that the nuclear transcriptome will be a useful reflection of the cytoplasmic transcriptome. The nuclear transcriptome differs in that it contains many pre-mRNAs that are not represented in the cytoplasm. Nuclear transcripts may pass out of the nucleus relatively quickly into the cytoplasm, where they may then accumulate and persist over relatively long time periods. Complex regulatory processes exist to ensure that nuclear transcript synthesis maintains a steady-state cytoplasmic abundance, and generally greater cytoplasmic transcript half-lives mean that nuclear synthesis rates and steady-state concentrations are likely to be much lower (Timmers and Tora, 2018). Especially of interest amongst cytoplasmic transcripts are those with roles in presynaptic terminals. Nuclear transcripts may not give much or any information regarding these as both their abundance and their rate of translation to protein could be greater within synaptic terminals than elsewhere in the cytoplasm or nucleus (Jung et al., 2014; Jung and Holt, 2011; Kim and Jung, 2020).
All single-cell methods need to take into account the likelihood that some cells or nuclei or transcripts may be lost in processing or will have processing-related abundance changes and these losses and changes are likely to be selective rather than general. We have tried to approach this by comparing deconvoluted transcriptomes from isolated whole cells with those from adjacent bulk tissue homogenates. One of the more remarkable results, reported by ourselves and at least one other group, is that the cell isolation and/or sorting protocols can cause significant artifactual upregulation of microglial transcripts and that these may mask in vivo group differences. (Serrano et al., 2021; Kang et al., 2018; Kang et al., 2017). It is possible that this effect may explain the lack of microglial transcript changes in this interesting report by Leng et al.
All methods have strengths and weaknesses and so we believe it is important to constantly cross-check results across methodologies. We encourage readers to compare the results from Leng et al. with our group’s published results on laser-captured tangle-bearing and non-tangle-bearing neurons (Dunckley et al., 2006), as well the cited reference (Liang et al., 2008) and several others (Liang et al., 2007; Liang et al., 2010; Liang et al., 2008; Liang et al., 2007; Mastroeni et al., 2018; Stamper et al., 2008).
The human brain material was obtained from autopsies with a very wide range in postmortem intervals (PMI), from 12 to 50 hours. Longer PMIs will result in progressive loss of selective transcripts that may be tissue, region, gene, and even genotype-dependent (Zhu et al., 2017; Birdsill et al., 2011; Walker et al., 2016). Did the authors attempt to determine, within their cases, whether PMI influenced the abundance of any transcripts, and, in particular, any of the transcripts identified as marking selectively vulnerable neurons?
Although the authors examined brain tissue from several Braak stages of AD, the subjects had various combinations of pathology, including absence of tau and Aβ, presence of tau but not Aβ, presence of Aβ but not tau, and presence of both. To isolate the changes relating to these two major types of AD pathology, future studies should look for expression changes associated with disease progression involving only Aβ or only tau, or in a group with both changes present from the start.
Additionally, it is well known that AD is most commonly complicated by one or more other major age-related molecular or vascular pathologies (Beach and Malek-Ahmadi, 2020) that have significant clinical effects. These would presumably have significant effects on gene expression as well, but so far there have not been concerted efforts to incorporate the full complexity inherent to the aging brain.
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