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Alzheimer’s disease (AD) is characterized by the selective vulnerability of specific neuronal populations, the molecular signatures of which are largely unknown. To identify and characterize selectively vulnerable neuronal populations, we used single-nucleus RNA sequencing to profile the caudal entorhinal cortex and the superior frontal gyrus – brain regions where neurofibrillary inclusions and neuronal loss occur early and late in AD, respectively – from postmortem brains spanning the progression of AD-type tau neurofibrillary pathology. We identified RORB as a marker of selectively vulnerable excitatory neurons in the entorhinal cortex, and subsequently validated their depletion and selective susceptibility to neurofibrillary inclusions during disease progression using quantitative neuropathological methods. We also discovered an astrocyte subpopulation, likely representing reactive astrocytes, characterized by decreased expression of genes involved in homeostatic functions. Our characterization of selectively vulnerable neurons in AD paves the way for future mechanistic studies of selective vulnerability and potential therapeutic strategies for enhancing neuronal resilience.

Highlights d MeCP2 represses transcription of highly methylated long genes through NCoR d Direct measurements of transcriptional initiation and elongation rates in the mouse brain d MeCP2 reduces transcriptional initiation, not elongation, of highly methylated long genes d Gene body-TSS contacts position distal MeCP2 molecules at the TSS

24Primary somatosensory neurons are specialized to transmit specific types of sensory 25 information through differences in cell size, myelination, and the expression of distinct 26 receptors and ion channels, which together define their transcriptional and functional 27 identity. By transcriptionally profiling sensory ganglia at single-cell resolution, we find that 28 different somatosensory neuronal subtypes undergo a remarkably consistent and 29 dramatic transcriptional response to peripheral nerve injury that both promotes axonal 30 regeneration and suppresses cell identity. Successful axonal regeneration leads to a 31 restoration of neuronal cell identity and the deactivation of the growth program. This 32 injury-induced transcriptional reprogramming requires Atf3, a transcription factor which is 33 induced rapidly after injury and is necessary for axonal regeneration and functional 34 recovery. While Atf3 and other injury-induced transcription factors are known for their role 35 in reprogramming cell fate, their function in mature neurons is likely to facilitate major 36 adaptive changes in cell function in response to damaging environmental stimuli. 37 38

In females with X-linked genetic disorders, wild-type and mutant cells coexist within brain tissue because of X-chromosome inactivation, posing challenges for interpreting the effects of X-linked mutant alleles on gene expression. We present a single-nucleus RNA sequencing approach that resolves mosaicism by using SNPs in genes expressed in cis with the X-linked mutation to determine which nuclei express the mutant allele even when the mutant gene is not detected. This approach enables gene expression comparisons between mutant and wild-type cells within the same individual, eliminating variability introduced by comparisons to controls with different genetic backgrounds. We apply this approach to mosaic female mouse models and humans with Rett syndrome, an X-linked neurodevelopmental disorder caused by mutations in the methyl-DNA-binding protein MECP2 and observe that cell-type-specific DNA methylation predicts the degree of gene up-regulation in MECP2 -mutant neurons. This approach can be broadly applied to study gene expression in mosaic X-linked disorders.

Graphical Abstract Highlights d Multimodal analysis differentiates cells beyond transcriptomic classification d Single-cell analysis links stimulus-induced calcium elevations to transcriptomes d Cell-type-specific responses to neurotransmitters are associated with maturation d Serotonergic signaling in human radial glia promotes radial fiber formation

Alzheimer's disease (AD) is characterized by the selective vulnerability of specific neuronal populations, the molecular signatures of which are largely unknown. To identify and characterize selectively vulnerable neuronal populations, we used single-nucleus RNA sequencing to profile the caudal entorhinal cortex and the superior frontal gyrus -brain regions where neurofibrillary inclusions and neuronal loss occur early and late in AD, respectively -from individuals spanning the neuropathological progression of AD. We identified RORB as a marker of selectively vulnerable excitatory neurons in the entorhinal cortex, and subsequently validated their depletion and selective susceptibility to neurofibrillary inclusions during disease progression using quantitative neuropathological methods. We also discovered an astrocyte subpopulation, likely representing reactive astrocytes, characterized by decreased expression of genes involved in homeostatic functions. Our characterization of selectively vulnerable neurons in AD paves the way for future mechanistic studies of selective vulnerability and potential therapeutic strategies for enhancing neuronal resilience. MAIN TEXTSelective vulnerability is a fundamental feature of neurodegenerative diseases, in which different neuronal populations show a gradient of susceptibility to degeneration 1, 2 . Selective vulnerability at the network level has been extensively explored in Alzheimer's disease (AD) 3-5 , currently the leading cause of dementia and lacking in effective therapies. However, little is known about the mechanisms underlying selective vulnerability at the cellular level in AD, which could provide insight into disease mechanisms and lead to therapeutic strategies.The entorhinal cortex (EC), an allocortex, is one of the first cortical brain regions to exhibit neuronal loss in AD 6 . Neurons in the external EC layers, especially in layer II (also known as alpha clusters of the lamina principalis externa, abbreviated "Pre-alpha") 7 , accumulate taupositive neurofibrillary changes and die early on in the course of AD 8-13 . However, these selectively vulnerable neurons have yet to be characterized extensively at the molecular level. Furthermore, it is unknown whether there are differences in vulnerability among subpopulations of these neurons. Although rodent models of AD have offered some insights [14][15][16] , the human brain has unique features with regard to cellular physiology, composition and aging [17][18][19] , limiting the extrapoloation of findings from animal models to address selective vulnerability.Previous studies have combined laser capture microdissection with microarray analysis of gene expression 20, 21 to characterize EC neurons in AD, but focused on disease-related changes in gene expression, rather than selective vulnerability. More recently, single-nucleus RNA-sequencing (snRNA-seq) has enabled large-scale characterization of transcriptomic profiles of individual cells from post-mortem human brain tissue 22, 23 . However, snRNA-seq studies of AD p...

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