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SummaryThe sheer complexity of the brain has complicated our ability to understand its cellular mechanisms in health and disease. Genome-wide association studies have uncovered genetic variants associated with specific neurological phenotypes and diseases. In addition, single-cell transcriptomics have provided molecular descriptions of specific brain cell types and the changes they undergo during disease. Although these approaches provide a giant leap forward towards understanding how genetic variation can lead to functional changes in the brain, they do not establish molecular mechanisms. To address this need, we developed a 3D co-culture system termed iAssembloids (induced multi-lineage assembloids) that enables the rapid generation of homogenous neuron-glia spheroids. We characterize these iAssembloids with immunohistochemistry and single-cell transcriptomics and combine them with large-scale CRISPRi-based screens. In our first application, we ask how glial and neuronal cells interact to control neuronal death and survival. Our CRISPRi-based screens identified that GSK3β inhibits the protective NRF2-mediated oxidative stress response in the presence of reactive oxygen species elicited by high neuronal activity, which was not previously found in 2D monoculture neuron screens. We also apply the platform to investigate the role of APOE-χ4, a risk variant for Alzheimer’s Disease, in its effect on neuronal survival. This platform expands the toolbox for the unbiased identification of mechanisms of cell-cell interactions in brain health and disease.

Astrocytes become reactive in response to insults to the central nervous system by adopting context-specific cellular signatures and outputs, but a systematic understanding of the underlying molecular mechanisms is lacking. In this study, we developed CRISPR interference screening in human induced pluripotent stem cell-derived astrocytes coupled to single-cell transcriptomics to systematically interrogate cytokine-induced inflammatory astrocyte reactivity. We found that autocrine-paracrine IL-6 and interferon signaling downstream of canonical NF-κB activation drove two distinct inflammatory reactive signatures, one promoted by STAT3 and the other inhibited by STAT3. These signatures overlapped with those observed in other experimental contexts, including mouse models, and their markers were upregulated in human brains in Alzheimer's disease and hypoxic-ischemic encephalopathy. Furthermore, we validated that markers of these signatures were regulated by STAT3 in vivo using a mouse model of neuroinflammation. These results and the platform that we established have the potential to guide the development of therapeutics to selectively modulate different aspects of inflammatory astrocyte reactivity.

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.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.

In response to central nervous system injury or disease, astrocytes become reactive, adopting context-dependent states and functional outputs. Certain inflammatory insults induce reactive astrocytes that lose homeostatic functions and gain harmful outputs through cellular pathways that are not fully understood. Here, we combined single-cell transcriptomics with CRISPRi screening in human iPSC-derived astrocytes to systematically interrogate inflammatory astrocyte reactivity. We found that autocrine-paracrine IL-6 and interferon signaling downstream of canonical NF-κB activation drove two distinct inflammatory reactive signatures – one promoted by and the other inhibited by STAT3. These signatures overlapped with those observed in other experimental contexts, including mouse models, and their markers were upregulated in the human brain in Alzheimer’s disease and hypoxic ischemic encephalopathy. Furthermore, we validated that these signatures were regulated by Stat3in vivo.These results and the platform we established have the potential to guide the development of therapeutics to selectively modulate different aspects of inflammatory astrocyte reactivity.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate iPSC lines and deeply characterised their genetic properties using whole genome sequencing, their genomic stability upon CRISPR/Cas9-based gene editing, and their phenotypic properties including differentiation to commonly-used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and hundreds of its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.SummaryThe authors of this collaborative study deeply characterized human induced pluripotent stem cell (iPSC) lines to rationally select a clonally-derived cell line that performs well across multiple modalities. KOLF2.1J was identified as a candidate reference cell line based on single-cell analysis of its gene expression in the pluripotent state, whole genome sequencing, genomic stability after highly efficient CRISPR-mediated gene editing, integrity of the p53 pathway, and the efficiency with which it differentiated into multiple target cell populations. Since it is deeply characterized and can be readily acquired, KOLF2.1J is an attractive reference cell line for groups working with iPSCs.Graphical abstract

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 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.

This protocol describes thedifferentiation of iPSCs with stably integrated doxycycline-inducible Ngn2 (such as i3Ns).

Mutations in the methyl-DNA-binding repressor protein MeCP2 cause the devastating neurodevelopmental disorder Rett syndrome. It has been challenging to understand how MeCP2 regulates transcription because MeCP2 binds broadly across the genome, and MeCP2 mutations are associated with widespread small-magnitude changes in neuronal gene expression. We demonstrate here that MeCP2 represses nascent RNA transcription of highly methylated long genes in the brain through its interaction with the NCoR co-repressor complex. By measuring the rates of transcriptional initiation and elongation directly in the brain, we find that MeCP2 has no measurable effect on transcriptional elongation, but instead represses the rate at which Pol II initiates transcription of highly methylated long genes. These findings suggest a new model of MeCP2 function in which MeCP2 binds broadly across highly methylated regions of DNA, but acts at transcription start sites to attenuate transcriptional initiation.

Neurological dysfunction following epileptic seizures is a well-recognized phenomenon. Several potential mechanisms have been suggested to explain postictal dysfunction, with alteration in cerebral blood flow being one possibility. These vascular disturbances may be long lasting and localized to brain areas involved in seizure generation and propagation, as supported by both animal and human studies. Therefore, measuring perfusion changes in the postictal period may help localize the seizure onset zone. Arterial spin labelling is a non-invasive, rapid and reproducible magnetic resonance imaging technique that measures cerebral perfusion. To this end, we measured postictal perfusion in patients with drug resistant focal epilepsy who were admitted to our seizure-monitoring unit for presurgical evaluation. Twenty-one patients were prospectively recruited and underwent arterial spin labelling scanning within 90 min of a habitual seizure. Patients also underwent a similar scan in the interictal period, after they were seizure-free for at least 24 h. The acquired scans were subtracted to identify the areas of significant postictal hypoperfusion. The location of the maximal hypoperfusion was compared to the presumed seizure onset zone to assess for concordance. Also, the localizing value of this technique was compared to other structural and functional imaging modalities. Postictal perfusion reductions of >15 units (ml/100 g/l) were seen in 15/21 patients (71.4%). In 12/15 (80%) of these patients, the location of the hypoperfusion was partially or fully concordant with the location of the presumed seizure onset zone. This technique compared favourably to other neuroimaging modalities, being similar or superior to structural magnetic resonance imaging in 52% of cases, ictal single-photon emission computed tomography in 60% of cases and interictal positron emission tomography in 71% of cases. Better arterial spin labelling results were obtained in patients in whom the seizure onset zone was discernible based on non-invasive data. Thus, this technique is a safe, non-invasive and relatively inexpensive tool to detect postictal hypoperfusion that may provide useful data to localize the seizure onset zone. This technique may be incorporated into the battery of conventional investigations for presurgical evaluation of patients with drug resistant focal epilepsy.

Primary somatosensory neurons are specialized to transmit specific types of sensory information through differences in cell size, myelination, and the expression of distinct receptors and ion channels, which together define their transcriptional and functional identity. By profiling sensory ganglia at single-cell resolution, we find that the all somatosensory neuronal subtypes undergo a similar transcriptional response to peripheral nerve injury that both promotes axonal regeneration and suppresses cell identity. This transcriptional reprogramming, which is not observed in non-neuronal cells, resolves over a similar time course as target reinnervation and is associated with the restoration of original cell identity. Injury-induced transcriptional reprogramming requires ATF3, a transcription factor which is induced rapidly after injury and necessary for axonal regeneration and functional recovery. Our findings suggest that transcription factors induced early after peripheral nerve injury likely confer the cellular plasticity required for sensory neurons to transform into a regenerative state.

This protocol describes thedifferentiation of iPSCs with stably integrated doxycycline-inducible Ngn2 (such as i3Ns).

ObjectiveSince the strongest risk factor for sudden unexpected death in epilepsy (SUDEP) is frequent bilateral tonic-clonic seizures (BTCS), our aim was to determine whether postictal hypoperfusion in brainstem respiratory centers (BRCs) is more common following tonic-clonic seizures.MethodsWe studied 21 patients with focal epilepsies who underwent perfusion imaging with arterial spin labeling MRI. Subtraction maps of cerebral blood flow were obtained from the postictal and baseline scans. We identified 6 regions of interest in the brainstem that contain key BRCs. Patients were considered to have postictal BRC hypoperfusion if any of the 6 regions of interest were significantly hypoperfused.ResultsAll 6 patients who experienced BTCS during the study had significant clusters of postictal hypoperfusion in BRCs compared to 7 who had focal impaired awareness seizures (7/15). The association between seizure type studied and the presence of BRC hypoperfusion was significant. Duration of epilepsy and frequency of BTCS were not associated with postictal brainstem hypoperfusion despite also being associated with risk for SUDEP.ConclusionPostictal hypoperfusion in brainstem respiratory centers occurs more often following BTCS than other seizure types, providing a possible explanation for the increased risk of SUDEP in patients who regularly experience BTCS.

Arterial spin labeling (ASL) MRI can provide seizure onset zone (SOZ) localizing information in up to 80% of patients. Clinical implementation of this technique is limited by the need to obtain two scans per patient: a postictal scan that is subtracted from an interictal scan. We aimed to determine whether it is possible to limit the number of ASL scans to one per patient by comparing patient postictal ASL scans to baseline scans of 100 healthy controls. Eighteen patients aged 20-55 years underwent ASL MRI

In the developing human neocortex, progenitor cells generate diverse cell types prenatally. Progenitor cells and newborn neurons respond to signaling cues, including neurotransmitters. While single-cell RNA sequencing has revealed cellular diversity, physiological heterogeneity has yet to be mapped onto these developing and diverse cell types. By combining measurements of intracellular Ca2+ elevations in response to neurotransmitter receptor agonists and RNA sequencing of the same single cells, we show that Ca2+ responses are cell-type-specific and change dynamically with lineage progression. Physiological response properties predict molecular cell identity and additionally reveal diversitynot captured by single-cell transcriptomics. We find that the serotonin receptor HTR2A selectively activates radial glia cells in the developing human, butnot mouse, neocortex, and inhibiting HTR2A receptorsin human radial glia disrupts the radial glial scaffold. We show highly specific neurotransmittersignaling during neurogenesis in the developing human neocortex and highlight evolutionarilydivergent mechanisms of physiological signaling.

Background We previously showed that CT perfusion (CTP) and arterial spin labelled (ASL) MRI can localize the seizure onset zone in humans via postictal perfusion patterns. As a step towards improving the feasibility/ease of collecting postictal CBF data, we determined whether EEG electrodes need to be removed for CTP data collection and whether a cross-modality comparison between baseline ASL and postictal CTP data is possible. New method Five patients with epilepsy underwent postictal CTP scanning. Three patients had an interictal ASL scan; one patient had both an ASL and CTP interictal scan. Postictal CTP maps were quantitatively compared to 1) ASL maps averaged from 100 healthy controls, 2) each patient’s baseline ASL map and 3) each patient’s baseline CTP map. To assess for electrode artifacts, a phantom and one patient underwent CTP scanning with EEG electrodes in place. The acquired scans were assessed for artifacts and for postictal hypoperfusion. Results Focal postictal hypoperfusion was observable only in intra-modality comparisons (CTP to CTP) and not in cross-modality comparisons (CTP to ASL). EEG electrodes produced streaking artifact that decreased image quality and precluded quantitative analysis. Comparison with existing methods(s) An intra-modality comparison of baseline CTP to postictal CTP was the only comparison method that showed localized hypoperfusion. Conclusions Quantitative comparison between postictal CTP and baseline ASL scans is not feasible. Postictal hypoperfusion can be detected by CTP only when two CTP scans are collected and when metallic EEG electrodes are removed.

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 single-nucleotide polymorphisms 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 gene encoding the methyl-DNA-binding protein MECP2, and observe that cell-type-specific DNA methylation predicts the degree of gene upregulation in MECP2-mutant neurons. This approach can be broadly applied to study gene expression in mosaic X-linked disorders.