Toward a CRISPR understanding of gene function in human brain development.

Genome-wide association studies pinpoint genetic risk factors for neurodevelopmental disorders (NDDs), but the next challenge is to understand the mechanisms through which these genes affect brain development. Two recent CRISPR screens in human brain organoids1,2 interrogate the function of risk genes for autism spectrum disorder and other NDDs.

Activity-induced MeCP2 phosphorylation regulates retinogeniculate synapse refinement.

Mutations in MECP2 give rise to Rett syndrome (RTT), an X-linked neurodevelopmental disorder that results in broad cognitive impairments in females. While the exact etiology of RTT symptoms remains unknown, one possible explanation for its clinical presentation is that loss of MECP2 causes miswiring of neural circuits due to defects in the brain's capacity to respond to changes in neuronal activity and sensory experience. Here, we show that MeCP2 is phosphorylated at four residues in the mouse brain (S86, S274, T308, and S421) in response to neuronal activity, and we generate a quadruple knock-in (QKI) mouse line in which all four activity-dependent sites are mutated to alanines to prevent phosphorylation. QKI mice do not display overt RTT phenotypes or detectable gene expression changes in two brain regions. However, electrophysiological recordings from the retinogeniculate synapse of QKI mice reveal that while synapse elimination is initially normal at P14, it is significantly compromised at P20. Notably, this phenotype is distinct from the synapse refinement defect previously reported for Mecp2 null mice, where synapses initially refine but then regress after the third postnatal week. We thus propose a model in which activity-induced phosphorylation of MeCP2 is critical for the proper timing of retinogeniculate synapse maturation specifically during the early postnatal period.

Activity-Induced MeCP2 Phosphorylation Regulates Retinogeniculate Synapse Refinement.

Mutations in MECP2 give rise to Rett syndrome (RTT), an X-linked neurodevelopmental disorder that results in broad cognitive impairments in females. While the exact etiology of RTT symptoms remains unknown, one possible explanation for its clinical presentation is that loss of MeCP2 causes miswiring of neural circuits due to defects in the brain's capacity to respond to changes in neuronal activity and sensory experience. Here we show that MeCP2 is phosphorylated at four residues in the brain (S86, S274, T308, and S421) in response to neuronal activity, and we generate a quadruple knock-in (QKI) mouse line in which all four activity-dependent sites are mutated to alanines to prevent phosphorylation. QKI mice do not display overt RTT phenotypes or detectable gene expression changes in two brain regions. However, electrophysiological recordings from the retinogeniculate synapse of QKI mice reveal that while synapse elimination is initially normal at P14, it is significantly compromised at P20. Notably, this phenotype is distinct from that previously reported for Mecp2 null mice, where synapses initially refine but then regress after the third postnatal week. We thus propose a model in which activity-induced phosphorylation of MeCP2 is critical for the proper timing of retinogeniculate synapse maturation specifically during the early postnatal period. SIGNIFICANCE STATEMENT: Rett syndrome (RTT) is an X-linked neurodevelopmental disorder that predominantly affects girls. RTT is caused by loss of function mutations in a single gene MeCP2. Girls with RTT develop normally during their first year of life, but then experience neurological abnormalities including breathing and movement difficulties, loss of speech, and seizures. This study investigates the function of the MeCP2 protein in the brain, and how MeCP2 activity is modulated by sensory experience in early life. Evidence is presented that sensory experience affects MeCP2 function, and that this is required for synaptic pruning in the brain. These findings provide insight into MeCP2 function, and clues as to what goes awry in the brain when the function of MeCP2 is disrupted.

Harnessing the power of collective intelligence in dentistry: a pilot study in Victoria, Australia.

BACKGROUND: In many dental settings, diagnosis and treatment planning is the responsibility of a single clinician, and this process is inevitably influenced by the clinician's own heuristics and biases. Our aim was to test whether collective intelligence increases the accuracy of individual diagnoses and treatment plans, and whether such systems have potential to improve patient outcomes in a dental setting. METHODS: This pilot project was carried out to assess the feasibility of the protocol and appropriateness of the study design. We used a questionnaire survey and pre-post study design in which dental practitioners were involved in the diagnosis and treatment planning of two simulated cases. Participants were provided the opportunity to amend their original diagnosis/treatment decisions after viewing a consensus report made to simulate a collaborative setting. RESULTS: Around half (55%, n = 17) of the respondents worked in group private practices, however most practitioners (74%, n = 23) did not collaborate when planning treatment. Overall, the average practitioners' self-confidence score in managing different dental disciplines was 7.22 (s.d. 2.20) on a 1-10 scale. Practitioners tended to change their mind after viewing the consensus response, particularly for the complex case compared to the simple case (61.5% vs 38.5%, respectively). Practitioners' confidence ratings were also significantly higher (p < 0.05) after viewing the consensus for complex case. CONCLUSION: Our pilot study shows that collective intelligence in the form of peers' opinion can lead to modifications in diagnosis and treatment planning by dentists. Our results lay the foundations for larger scale investigations on whether peer collaboration can improve diagnostic accuracy, treatment planning and, ultimately, oral health outcomes.

CRISPRi-based screens in iAssembloids to elucidate neuron-glia interactions.

The 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 GSK3ß 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-ε4, 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.

Treatment outcomes and their determinants of IgG4-related ophthalmic disease: a territory-wide cohort study.

BACKGROUND: Oral corticosteroid remains the first-line treatment of IgG4-related ophthalmic disease, but steroid-dependence is common and serious. Factors associated with steroid dependence and relapse have to be further explored. STUDY POPULATION: A city-wide, biopsy-proven, Chinese cohort. METHODS: Retrospective, masked review of medical records, orbital images and histopathology reports. RESULTS: There were 101 patients with at least 24-month follow-up. Up to 82% (82/101) received oral corticosteroid as first-line treatments, and 7 of them received also concomitant steroid-sparing agents (SSA)/biological agents as primary treatment. There was 61% (50/82) of patients required long-term corticosteroid (alone=23, with SSA=27) after 1.9±0.7 (range 1-5) relapses. When compared with the 21% (17/82) of patients who tapered corticosteroid successfully for 24 months, steroid dependence was associated with elevated baseline serum IgG4 level (94% vs 65%, p<0.01) and Mikulicz syndrome (46% vs 18%, p<0.05). Up to 13% (11/82) of patients tolerated residual disease after tapering off corticosteroid. There was 17% (17/101) of patients did not require any medications after biopsies. They were more likely to have debulking surgeries (71% vs 40%, p<0.05), discrete orbital lesions (65% vs 26%, p<0.05), normal baseline serum IgG4 level (24% vs 6%, p<0.05) and no Mikulicz syndrome (94% vs 61%, p<0.05). CONCLUSION: In this cohort, 60% of patients required long-term maintenance oral corticosteroid. Elevated pretreatment serum IgG4 level and Mikulicz syndrome were associated with steroid dependence. Debulking surgery is an alternative for a subgroup of patients with discrete orbital lesions, normal baseline IgG4 level and no Mikulicz syndrome.

Malignancies in Immunoglobulin G4-related ophthalmic disease.

PURPOSE: Clinical phenotypes in Immunoglobulin G4-related disease (IgG4-RD) according to the patterns of affecting organs have different risks of malignancies. We attempt to determine the association of malignancies with IgG4-related ophthalmic disease (IgG4-ROD). DESIGN: Retrospective cohort study. METHODS: Review of medical records, orbital images and histopathology reports in a territory-wide cohort of biopsy proven IgG4-ROD patients from 2005-2019. FINDINGS: Among 122 patients who had biopsies taken from adnexal lesions including lacrimal glands (n = 108), orbital mass (n = 30), infiltrated orbital fat (n = 10), conjunctiva (n = 2) or extraocular muscles (n = 3), 13% (16/122) developed malignancies over 73 ± 48months' follow-up. There were 9 cases of ocular adnexal lymphoma (OAL) and 7 extra-orbital malignancies. Compared with the general population, the incidence of OAL was significantly higher (standardized incidence ratios, SIRs = 10.0, 95%CI = 4.5-17.6) while that of extra-orbital malignancies was similar. The SIRs was highest within the first year (SIR = 46.7, 95%CI = 18.5-87.6) when 7 OAL were concomitantly diagnosed. Patients who developed OAL or extra-orbital malignancies were older than other patients at IgG4-ROD diagnosis (64.9 ± 7.1, 68.3 ± 8.5 versus 55.2 ± 15.0 years, P < 0.05). Asymmetric lacrimal gland enlargement (78% versus 13%), lack of frontal (0% versus 12%) or infraorbital nerve enlargement (0% versus 36%) were associated with OAL (all P < 0.05). Pre-treatment serum IgG4 level or extra-orbital IgG4-RD involvement was similar among patients with or without malignancies. CONCLUSION: In this biopsy-proven IgG4-ROD cohort, 7% developed OAL which was 10 times higher than the general population. Patients with asymmetric lacrimal gland enlargement or without trigeminal nerves involvement radiologically were associated with OAL.

CRISPRi screens in human iPSC-derived astrocytes elucidate regulators of distinct inflammatory reactive states.

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.

Radiological Determinants of Complicated Immunoglobulin G4-Related Ophthalmic Disease: A Territory-Wide Cohort Study.

PURPOSE: To evaluate the presenting radiological features of immunoglobulin G4-related ophthalmic disease (IgG4-ROD) and their associations with IgG4-related optic neuropathy (IgG4-RON), and IgG4-related ocular adnexal lymphoma (IgG4-ROL). METHODS: A territory-wide, biopsy-proven, Chinese cohort. Masked review of orbital images, medical records, and histopathology reports. RESULTS: A total of 115 (94%) of the 122 patients in our cohort had preoperative orbital images (computed tomography=105, magnetic resonance imaging=40). Among them, 103/115 (90%) showed enlarged lacrimal glands, and 91 (88%) were bilateral. Nerve enlargement was observed: infraorbital in 31/115 (27%) patients and frontal in 17/115 (15%), 10 and 9 being bilateral, respectively. At least 1 or more extraocular muscle (EOM) enlargement was found in 41/115 (37%) patients, bilaterally in 20. Lateral rectus occurred in 30 (73%) of these 41 EOM patients and inferior rectus in 28 (68%). Two adjacent EOMs (inferior and lateral recti in 11 patients, inferior and medial recti in 7 patients) or multiple EOMs (at least 3) were enlarged in 23/41 (56%) and 13/41 (32%) of the patients, respectively. Intraconal lesions (67% vs 11%, P<0.05), infraorbital (83% vs 23%, P<0.005), or frontal (50% vs 15%, P<0.05) nerve enlargement was significantly associated with IgG4-RON (6 patients) by univariate analyses. Asymmetric lacrimal gland enlargement and discrete orbital mass (both P<0.05) were associated with IgG4-ROL (9 patients) by multivariate analyses. CONCLUSIONS: In this IgG4-ROD cohort, most patients had bilateral enlarged lacrimal glands, and the lateral rectus is the most frequently involved EOM. For the first time, unique radiological patterns associated with the development of IgG4-RON and IgG4-ROL are found.

Adult neurogenesis in crayfish: Identity and regulation of neural progenitors produced by the immune system.

Adult-born neurons are incorporated into brain circuits in the crayfish Procambarus clarkii, as in many vertebrate and invertebrate species. Adult neurogenesis depends on several conserved features, including the presence of neurogenic niches housing progenitor cells and the expansion, migration, and differentiation of their daughters, the neural precursor cells. However, in contrast to mammalian species, the progenitors initiating the neurogenic lineage in P. clarkii do not undergo long-term self-renewal. A central question is the mode of replenishment of these cells. Experiments have shown that hemocytes generated by the immune system, and not other cell types, are attracted to and incorporated into the niche. The present studies highlight the interdependency of the immune and nervous systems in the generation of adult-born neurons, by demonstrating that hyaline hemocytes are the probable neural progenitor cells, and that serotonin and the cytokine astakine 1 regulate both immune function and adult neurogenesis.

Molecular characterization of selectively vulnerable neurons in Alzheimer's disease.

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.

Willingness to pay for cataract surgery in baiyin district, northwestern China.

PURPOSE: To investigate willingness to pay for cataract surgery, and its associations, in Northwestern China. METHODS: Four hundred thirty-eight persons aged 50 years and above, diagnosed with cataract indicated for surgery, identified in an outreach screening program were included. Subjects were offered a willingness-to-pay interview for the maximal amount that the subjects would be willing to pay for a cataract surgery. Age, gender, literacy, education level, occupation, and annual household income were recorded. RESULTS: Among 328 (74.9%) subjects who completed the interview, 197 (60.1%) participants were willing to pay something for the cataract surgery (mean, 902.9 ± 856.7 renminbi[RMB], [US$ 145 ± 137]; median, 500RMB, US$ 78). Individuals with presenting visual acuity (PVA) in the worse eye ≤6/60 (OR: 2.1, 95% CI: 1.3-3.2) and a high annual household incomes (OR: 2.0, 95% CI: 0.9-4.6) were likely to be willing to pay for the surgery, as revealed in the regression models. Willingness to pay any amount for cataract surgery was more likely among literate persons (OR: 1.5, 95% CI: 1.0-2.4) and persons with non-agricultural occupation (OR: 1.8, 95% CI: 1.0-3.2). CONCLUSIONS: The amount that subjects were willing to pay is significantly less than the current cost of cataract surgery (5000 RMB, US$320) in the area. Providing low-cost cataract surgery to patients in a financially sustainable manner is important to increase uptake of cataract surgery among rural residents in Northwest China.

Transcriptional Reprogramming of Distinct Peripheral Sensory Neuron Subtypes after Axonal Injury.

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 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 that 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 confer the cellular plasticity required for sensory neurons to transform into a regenerative state.

Postictal brainstem hypoperfusion and risk factors for sudden unexpected death in epilepsy.

OBJECTIVE: Since 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. METHODS: We 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. RESULTS: All 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. CONCLUSION: Postictal 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.

Localizing the seizure onset zone by comparing patient postictal hypoperfusion to healthy controls.

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 <90 min after a seizure and during the interictal period. Each postictal cerebral blood flow (CBF) map was statistically compared to average baseline CBF maps from 100 healthy controls (pvcASL; patient postictal CBF vs. control baseline CBF). The pvcASL maps were compared to subtraction ASL maps (sASL; patient baseline CBF minus patient postictal CBF). Postictal CBF reductions from pvcASL and sASL maps were seen in 17 of 18 (94.4%) and 14 of 18 (77.8%) patients, respectively. Maximal postictal hypoperfusion seen in pvcASL and sASL maps was concordant with the SOZ in 10 of 17 (59%) and 12 of 14 (86%) patients, respectively. In seven patients, both pvcASL and sASL maps showed similar results. In two patients, sASL showed no significant hypoperfusion, while pvcASL showed significant hypoperfusion concordant with the SOZ. We conclude that pvcASL is clinically useful and although it may have a lower overall concordance rate than sASL, pvcASL does provide localizing or lateralizing information for specific cases that would be otherwise missed through sASL.

MeCP2 Represses the Rate of Transcriptional Initiation of Highly Methylated Long Genes.

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.

Diagnostic Accuracy of Rapid Assessment of Avoidable Blindness: A Population-based Assessment.

OBJECTIVES: To evaluate the diagnostic accuracy of rapid assessment of avoidable blindness (RAAB). DESIGN: Population-based diagnostic accuracy study. METHODS: A total of 2145 (95.3%, 2145/2250) subjects aged 50 years and older who participated in the RAAB survey were included. All the recruited participants underwent ophthalmic examination according to the RAAB protocol and then were reexamined with instruments in a mobile eye clinic set up in a village center on the same day. Examination in the mobile clinic included standardized visual acuity (VA) tests using logMAR charts, refraction, slit-lamp biomicroscopy, and dilated fundal examination with a binocular indirect ophthalmoscope. Blindness and economic blindness were defined as VA in the better-seeing eye <3/60 and <6/60, respectively. Visual impairment (VI) was defined as VA <6/18 in the better eye. The primary cause of blindness and VI was defined according to the cause of VI in the participant's better eye. MAIN OUTCOME MEASURES: The sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and area under the curve (AUC) of receiver operating characteristics of RAAB for detection of blindness and the principal causes of VI. RESULTS: A total of 1816 subjects (84.7%), including 686 men (37.8%) and 1130 women (62.2%), underwent ophthalmic examination in the mobile eye clinic. The mean (±standard deviation) age was 64.4 ± 9.6 years. The sensitivities, specificities, AUC, PLR, and NLR of RAAB were 90.3%, 99.3%, 0.948, 124.0, and 0.10, respectively, for detection of blindness (presenting visual acuity, PVA <3/60); 89.5%, 98.7%, 0.940, 69.2, and 0.11, respectively, for detection of economic blindness (PVA <6/60); and 90.3%, 97.7%, 0.940, 38.7, and 0.10, respectively, for detection of VI (PVA <6/18). The sensitivities, specificities, AUC, PLR, and NLR were 90.5%, 98.1%, 0.943, 48.1, and 0.10; and 60.4%, 98.7%, 0.796, 46.4, and 0.40 for detection of VI (PVA <6/18) owing to cataract and refractive error, respectively. CONCLUSION: The diagnostic performances of RAAB were high for detecting the prevalence of blindness, VI, and VI owing to cataract.

Multimodal Single-Cell Analysis Reveals Physiological Maturation in the Developing Human Neocortex.

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 diversity not captured by single-cell transcriptomics. We find that the serotonin receptor HTR2A selectively activates radial glia cells in the developing human, but not mouse, neocortex, and inhibiting HTR2A receptors in human radial glia disrupts the radial glial scaffold. We show highly specific neurotransmitter signaling during neurogenesis in the developing human neocortex and highlight evolutionarily divergent mechanisms of physiological signaling.

Quantitatively detecting postictal hypoperfusion in patients with focal epilepsy using CT perfusion: Determining cross-modality comparisons and electrode artifacts.

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.

Characterization of human mosaic Rett syndrome brain tissue by single-nucleus RNA sequencing.

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.