21 Jan 2021

As Alzheimer’s tangle pathology progresses, neurodegeneration sweeps through the brain in a stereotypical fashion. But why do some neurons perish early on, while their neighbors persist until the bitter end? A study published January 11 in Nature Neuroscience addressed this question by tracing the gene-expression profiles of neurons in the brains of people who died in the early or late stages of the disease. Among myriad subpopulations of cells, the researchers zeroed in on subsets of excitatory neurons that express the transcription factor RORB as the first to succumb. Initially in the entorhinal cortex, and then later in the outer neocortex, excitatory neurons bearing this particular marker were selectively vulnerable to tau accumulation, and to death. The study, led by Lea Grinberg and Martin Kampmann, both at the University of California, San Francisco, also pegged a type of reactive astrocyte that may shirk its duties of protecting and nourishing neurons. In all, the findings hint at common mechanisms underlying selective vulnerability to AD pathogenesis in different regions of the brain.

“The identification and molecular characterization of selectively vulnerable neurons in AD is a very important contribution,” commented Hansruedi Mathys of the Massachusetts Institute of Technology. “This work will undoubtedly facilitate future studies to understand why these neurons are so vulnerable.”​

For decades in Alzheimer’s research, selective vulnerability has loomed as the big question, without much progress toward an answer. “In Alzheimer’s disease, which is so complex, selective cellular vulnerability is a key anchoring point for mechanistic discovery,” wrote Jessica Rexach of the University of California, Los Angeles, to Alzforum. “We must understand what differs between vulnerable and spared cells as disease progresses. By using highly curated neuropathological specimens, and validation across independent studies, this important work sets an exciting foundation for continued discovery.”

As documented by Braak staging, neurofibrillary tangles overtake the brain in a defined sequence, appearing in the entorhinal cortex in early disease and moving to the outer reaches of the cortex in the final stages. Neuronal death tracks closely behind. Grinberg, a neuropathologist who has examined 3,000 human brains over the years, was struck by the selectivity with which neurons succumb to the disease. “Why is it that one neuron dies when tau tangles appear in the region, while its neighbor survives?” she wondered. Past studies have attempted to define regional and morphological characteristics of vulnerable cells, but Grinberg wanted to dive deeper and define vulnerability at the molecular level (Aug 2016 news). To probe the underpinnings of neuronal weakness to AD pathology, Grinberg joined forces with Kampmann, a cell biologist who uses genetic tools to unearth disease mechanisms.

Co-first authors Kun Leng, Emmy Li, and colleagues started by procuring brain samples from people who had died at different stages AD. Their cohort included 10 men, all ApoE3 homozygotes, who lived to between 50 and 91 years of age. Three had neuropathology in Braak stage 0, four in stage 2, and three in stage 6. From each brain, the researchers sampled cells from the entorhinal cortex (EC) and the superior frontal gyrus (SFG). These two regions are typically inundated with tau tangles by Braak stage II and V, respectively.

Across all the samples, the researchers harvested more than 42,000 cells from the EC, and 63,000 from the SFG. Using single-nucleus RNA sequencing, they analyzed the gene-expression profiles of each cell. While previous studies already used single-nuclei approaches to compare the transcriptomes of brain cells from people with AD to those of healthy controls, the current study is unique in its analysis along disease progression (May 2019 news; Nov 2019 news). 

Who Dies First? Researchers sampled the gene-expression profiles of neurons in the entorhinal cortex (EC) and superior frontal gyrus (SFG), regions invaded by tangles at early and late stages, respectively. [Courtesy of Leng et al., Nature Neuroscience, 2020.]

The first order of business was to identify selectively vulnerable cell types—i.e., those that decreased in number as disease progressed. Using broad cell type markers at first, the researchers saw a drop in excitatory neurons in the EC starting at Braak stage 2, and later a dip in excitatory neurons in the SFG at stage 6. In contrast, numbers of inhibitory neurons did not drop as disease got worse.

Next, the researchers zoomed in on transcriptomic differences between excitatory neurons within the EC, grouping nine distinct subpopulations based on their gene-expression profiles. Of these, three subpopulations halved in number in Braak stage 2, pegging them as a selectively vulnerable population.

Rooting Out the Weak. In the EC, researchers detected nine subpopulations of excitatory neurons (left). Of those, s1/amber, s2/olive, and s4/turquoise shrank in Braak stage 2 (right). On right, cells are color-coded by donor. [Courtesy of Leng et al., Nature Neuroscience, 2020.]

What made these three subsets so susceptible? Two of them uniquely expressed three transcripts—RORB, CTC-340A15.2, and CTC-535M15.2. The latter two are noncoding transcripts of unknown function. RORB (RAR-related Orphan Receptor b) encodes a transcription factor that drives the development of layer IV cortical neurons, although it is also expressed in other cortical layers (Jabaudon et al., 2012; Oishi et al., 2016). Its role in the adult brain is not understood.

Compared to other excitatory neurons in the EC, the vulnerable cells were relatively flush with transcripts encoding axonal proteins and voltage-gated potassium channels, while they had few transcripts encoding synaptic signaling proteins.

What happened in the SFG, where tau tangles don’t appear until Braak stage 5? There, the researchers identified 11 subpopulations of excitatory neurons. Of those, two shrank toward Braak stage 6. Notably, these two vulnerable SFGs populations also expressed the same three transcripts that stood out in vulnerable excitatory neurons in the EC. At a broad transcriptome level, the vulnerable SFG neurons had more in common with the vulnerable neurons in the EC than they did with any other EC subpopulation. Kampmann emphasized that this does not mean that the vulnerable neurons in the EC are the same exact type as those in the SFG. In fact, there were many gene-expression differences between them. However, it does suggest that similar mechanisms might underlie their tendency to crumble once tangles come to town.

The researchers spotted similar subpopulations of neurons within other gene-expression datasets from postmortem brain samples reported from Li Huei Tsai’s lab at MIT and Rick Livesey’s group at University College London (Mathys et al., 2019; Marinaro et al., 2020). 

To validate their snRNA-Seq findings, the researchers used immunofluorescence to evaluate 26 separate postmortem brain samples with AD pathology spanning Braak stages 0 to 6. In contrast to the all-male, ApoE3-only cohort used for the snRNA-Seq analysis, this cohort included 10 women and four ApoE4 carriers. The researchers reported that in this sample, too, the proportion of RORB-expressing excitatory neurons in the EC waned with increasing Braak stages, while excitatory neurons devoid of RORB did not decline. Strikingly, the researchers detected accumulation of phospho-tau specifically in RORB+ neurons. Morphologically, RORB+ excitatory neurons came in different shapes and sizes, including both pyramidal neurons and large, multipolar neurons (see image below). Together, the findings suggested that RORB+ neurons are selectively vulnerable to tauopathy in AD.

Shapes and Sizes. Large multipolar (m1 and m3-m5) and a pyramidal (p1) neuron express RORB (green). Some also accumulate tangles (red). From layer II EC of a person with AD. [Courtesy of Leng et al., Nature Neuroscience, 2020.]

While they were at it, the researchers also investigated what genes glial cells were expressing. For astrocytes, they found four subpopulations in the EC, six in the SFG. In each region, at least one such group practically screamed with GFAP, a marker of reactive astrocytes. These cells expressed few markers of homeostatic astrocytes, suggesting they had ditched their neuroprotective persona for reactive ones. The paper does not include a detailed analysis of how these reactive populations changed with worsening disease.

For microglia, the researchers detected four subpopulations in the EC, and five in the SFG. However, they were unable to find the transcripts that have been associated with homeostatic or disease-associated microglia in other studies (Jun 2017 news; Sep 2017 news; May 2019 news). The researchers suggest that low-abundance transcripts may have been lost because they resided in the cytoplasm, and the study used only nuclei. A recent study found that snRNA-Seq misses transcripts that have been linked to AD and might be abundant in the cytosol (Oct 2020 news). 

In a comment to Alzforum, Arizona-based researchers Thomas Beach and Geidy Serrano of Banner Sun Health Research Institute in Sun City; Diego Mastroeni of Arizona State University in Tempe; and Matthew Huentelman of the Translational Genomics Research Institute in Phoenix, discussed the trade-offs of using single-cell versus nuclei for transcriptomics. On one hand, plucking intact, whole cells from postmortem brain is fraught with difficulty, and single-nuclei approaches may more closely reflect the proportion of cells types found in vivo. On the other hand, nuclear transcriptomes cannot account for the many transcripts that are exported to the cytoplasm, especially those awaiting translation in synaptic terminals. “All methods have strengths and weaknesses, and so we believe it is important to constantly cross-check results across methodologies,” they wrote.

Kampmann believes the study provides a detailed molecular handle on which types of cells are most vulnerable. It also tightens the correlation between tau and cell death, albeit without clinching causality. “What our study does say is that the same types of neurons most likely to accumulate tau are also those that die first,” he said.

In regard to RORB, Kampmann said that cell-culture studies will reveal whether this transcription factor causes vulnerability, or merely marks cells that are vulnerable for a different, as-yet-unknown reason. Using CRISPR and iPSC-based cell models, he plans to investigate the role of RORB and other genes in rendering neurons susceptible to death. Grinberg and Kampmann are also expanding their snRNA-Seq studies to samples from women and people with different ApoE genotypes, as well as different regions of the brain, including subcortical areas gripped by tau pathology even before the entorhinal cortex.—Jessica Shugart