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.

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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Aim: Tau truncation (tr-tau) by active caspase-6 (aCasp-6) generates tau fragments that may be toxic. Yet the relationship between aCasp-6, different forms of tr-tau and hyperphosphorylated tau (p-tau) accumulation in human brains with Alzheimer's disease (AD) and other tauopathies remains unclear.Methods: We generated two neoepitope monoclonal antibodies against tr-tau sites (D402 and D13) targeted by aCasp-6. Then, we used five-plex immunofluorescence to quantify the neuronal and astroglial burden of aCasp-6, tr-tau, p-tau and their cooccurrence in healthy controls, AD and primary tauopathies.Results: Casp-6 activation was strongest in AD and Pick's disease (PiD) but almost absent in 4-repeat (4R) tauopathies. In neurons, the tr-tau burden was much more abundant in AD and PiD than in 4R tauopathies and disproportionally higher when normalising by p-tau pathology. Tr-tau astrogliopathy was detected in low numbers in 4R tauopathies. Unexpectedly, about half of tr-tau positive neurons in AD and PiD lacked p-tau aggregates, a finding we confirmed using several p-tau antibodies.Conclusions: Early modulation of aCasp-6 to reduce tr-tau pathology is a promising therapeutic strategy for AD and PiD but is unlikely to benefit 4R tauopathies. The large percentage of tr-tau-positive neurons lacking p-tau suggests that many vulnerable neurons to tau pathology go undetected when using conventional p-tau antibodies. Therapeutic strategies against tr-tau pathology could be necessary to modulate the extent of tau