Single-nucleus RNA sequencing demonstrates an autosomal dominant Alzheimer's disease profile and possible mechanisms of disease protection.

Highly penetrant autosomal dominant Alzheimer's disease (ADAD) comprises a distinct disease entity as compared to the far more prevalent form of AD in which common variants collectively contribute to risk. The downstream pathways that distinguish these AD forms in specific cell types have not been deeply explored. We compared single-nucleus transcriptomes among a set of 27 cases divided among PSEN1-E280A ADAD carriers, sporadic AD, and controls. Autophagy genes and chaperones clearly defined the PSEN1-E280A cases compared to sporadic AD. Spatial transcriptomics validated the activation of chaperone-mediated autophagy genes in PSEN1-E280A. The PSEN1-E280A case in which much of the brain was spared neurofibrillary pathology and harbored a homozygous APOE3-Christchurch variant revealed possible explanations for protection from AD pathology including overexpression of LRP1 in astrocytes, increased expression of FKBP1B, and decreased PSEN1 expression in neurons. The unique cellular responses in ADAD and sporadic AD require consideration when designing clinical trials.

Human tau mutations in cerebral organoids induce a progressive dyshomeostasis of cholesterol.

Mutations in the MAPT gene that encodes tau lead to frontotemporal dementia (FTD) with pathology evident in both cerebral neurons and glia. Human cerebral organoids (hCOs) from individuals harboring pathogenic tau mutations can reveal the earliest downstream effects on molecular pathways within a developmental context, generating interacting neurons and glia. We found that in hCOs carrying the V337M and R406W tau mutations, the cholesterol biosynthesis pathway in astrocytes was the top upregulated gene set compared with isogenic controls by single-cell RNA sequencing (scRNA-seq). The 15 upregulated genes included HMGCR, ACAT2, STARD4, LDLR, and SREBF2. This result was confirmed in a homozygous R406W mutant cell line by immunostaining and sterol measurements. Cholesterol abundance in the brain is tightly regulated by efflux and cholesterol biosynthetic enzyme levels in astrocytes, and dysregulation can cause aberrant phosphorylation of tau. Our findings suggest that cholesterol dyshomeostasis is an early event in the etiology of neurodegeneration caused by tau mutations.

Functional neuronal circuitry and oscillatory dynamics in human brain organoids.

Human brain organoids replicate much of the cellular diversity and developmental anatomy of the human brain. However, the physiology of neuronal circuits within organoids remains under-explored. With high-density CMOS microelectrode arrays and shank electrodes, we captured spontaneous extracellular activity from brain organoids derived from human induced pluripotent stem cells. We inferred functional connectivity from spike timing, revealing a large number of weak connections within a skeleton of significantly fewer strong connections. A benzodiazepine increased the uniformity of firing patterns and decreased the relative fraction of weakly connected edges. Our analysis of the local field potential demonstrate that brain organoids contain neuronal assemblies of sufficient size and functional connectivity to co-activate and generate field potentials from their collective transmembrane currents that phase-lock to spiking activity. These results point to the potential of brain organoids for the study of neuropsychiatric diseases, drug action, and the effects of external stimuli upon neuronal networks.

CREST, a Cas13-Based, Rugged, Equitable, Scalable Testing (CREST) for SARS-CoV-2 Detection in Patient Samples.

The COVID-19 pandemic has taken a devastating human toll worldwide. The development of impactful guidelines and measures for controlling the COVID-19 pandemic requires continuous and widespread testing of suspected cases and their contacts through accurate, accessible, and reliable methods for SARS-CoV-2 detection. Here we describe a CRISPR-Cas13-based method for the detection of SARS-CoV-2. The assay is called CREST (Cas13-based, rugged, equitable, scalable testing), and is specific, sensitive, and highly accessible. As such, CREST may provide a low-cost and dependable alternative for SARS-CoV-2 surveillance. © 2022 Wiley Periodicals LLC. Basic Protocol: Cas13-ased detection of SARS-CoV-2 genetic material using a real-time PCR detection system Alternate Protocol: Cas13-based detection of SARS-CoV-2 genetic material using a fluorescence viewer Support Protocol 1: LwaCas13a purification Support Protocol 2: In vitro transcription of synthetic targets.

Comparison of Severe Acute Respiratory Syndrome Coronavirus 2 Screening Using Reverse Transcriptase-Quantitative Polymerase Chain Reaction or CRISPR-Based Assays in Asymptomatic College Students.

Importance: The reopening of colleges and universities in the US during the coronavirus disease 2019 (COVID-19) pandemic is a significant public health challenge. The development of accessible and practical approaches for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in the college population is paramount for deploying recurrent surveillance testing as an essential strategy for virus detection, containment, and mitigation. Objective: To determine the prevalence of SARS-CoV-2 in asymptomatic participants in a university community by using CREST (Cas13-based, rugged, equitable, scalable testing), a CRISPR-based test developed for accessible and large-scale viral screening. Design, Setting, and Participants: For this cohort study, a total of 1808 asymptomatic participants were screened for SARS-CoV-2 using a CRISPR-based assay and a point-of-reference reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) test. Viral prevalence in self-collected oropharyngeal swab samples collected from May 28 to June 11, 2020, and from June 23 to July 2, 2020, was evaluated. Exposures: Testing for SARS-CoV-2. Main Outcomes and Measures: SARS-CoV-2 status, viral load, and demographic information of the study participants were collected. Results: Among the 1808 participants (mean [SD] age, 27.3 [11.0] years; 955 [52.8%] female), 732 underwent testing from May to early June (mean [SD] age, 28.4 [11.7] years; 392 [53.6%] female). All test results in this cohort were negative. In contrast, 1076 participants underwent testing from late June to early July (mean [SD] age, 26.6 [10.5] years; 563 [52.3%] female), with 9 positive results by RT-qPCR. Eight of these positive samples were detected by the CRISPR-based assay and confirmed by Clinical Laboratory Improvement Amendments-certified diagnostic testing. The mean (SD) age of the positive cases was 21.7 (3.3) years; all 8 individuals self-identified as students. These metrics showed that a CRISPR-based assay was effective at capturing positive SARS-CoV-2 cases in this student population. Notably, the viral loads detected in these asymptomatic cases resemble those seen in clinical samples, highlighting the potential of covert viral transmission. The shift in viral prevalence coincided with the relaxation of stay-at-home measures. Conclusions and Relevance: These findings reveal a shift in SARS-CoV-2 prevalence in a young and asymptomatic population and uncover the leading edge of a local outbreak that coincided with rising case counts in the surrounding county and the state of California. The concordance between CRISPR-based and RT-qPCR testing suggests that CRISPR-based assays are reliable and offer alternative options for surveillance testing and detection of SARS-CoV-2 outbreaks, as is required to resume operations in higher-education institutions in the US and abroad.

A Scalable, Easy-to-Deploy Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material.

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly trained personnel, and large upfront investment. Here, we showcase an orthogonal pipeline we call CREST (Cas13-based, rugged, equitable, scalable testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows a binary interpretation of results. CREST may provide a point-of-care solution to increase the distribution of COVID-19 surveillance.

LRP1 is a master regulator of tau uptake and spread.

The spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau has a central role in the pathogenesis of several forms of dementia known as tauopathies-including Alzheimer's disease, frontotemporal dementia and chronic traumatic encephalopathy1. Progression of these diseases is characterized by the sequential spread and deposition of protein aggregates in a predictable pattern that correlates with clinical severity2. This observation and complementary experimental studies3,4 have suggested that tau can spread in a prion-like manner, by passing to naive cells in which it templates misfolding and aggregation. However, although the propagation of tau has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein receptor-related protein 1 (LRP1) controls the endocytosis of tau and its subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and in induced pluripotent stem cell-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule-binding repeat region of tau. Furthermore, downregulation of LRP1 in an in vivo mouse model of tau spread was found to effectively reduce the propagation of tau between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain, and therefore a potential target for the treatment of diseases that involve tau spread and aggregation.

Control over single-cell distribution of G1 lengths by WNT governs pluripotency.

The link between single-cell variation and population-level fate choices lacks a mechanistic explanation despite extensive observations of gene expression and epigenetic variation among individual cells. Here, we found that single human embryonic stem cells (hESCs) have different and biased differentiation potentials toward either neuroectoderm or mesendoderm depending on their G1 lengths before the onset of differentiation. Single-cell variation in G1 length operates in a dynamic equilibrium that establishes a G1 length probability distribution for a population of hESCs and predicts differentiation outcome toward neuroectoderm or mesendoderm lineages. Although sister stem cells generally share G1 lengths, a variable proportion of cells have asymmetric G1 lengths, which maintains the population dispersion. Environmental Wingless-INT (WNT) levels can control the G1 length distribution, apparently as a means of priming the fate of hESC populations once they undergo differentiation. As a downstream mechanism, global 5-hydroxymethylcytosine levels are regulated by G1 length and thereby link G1 length to differentiation outcomes of hESCs. Overall, our findings suggest that intrapopulation heterogeneity in G1 length underlies the pluripotent differentiation potential of stem cell populations.

Action potential propagation recorded from single axonal arbors using multielectrode arrays.

We report the presence of co-occurring extracellular action potentials (eAPs) from cultured mouse hippocampal neurons among groups of planar electrodes on multielectrode arrays (MEAs). The invariant sequences of eAPs among coactive electrode groups, repeated co-occurrences, and short interelectrode latencies are consistent with action potential propagation in unmyelinated axons. Repeated eAP codetection by multiple electrodes was widespread in all our data records. Codetection of eAPs confirms they result from the same neuron and allows these eAPs to be isolated from all other spikes independently of spike sorting algorithms. We averaged co-occurring events and revealed additional electrodes with eAPs that would otherwise be below detection threshold. We used these eAP cohorts to explore the temperature sensitivity of action potential propagation and the relationship between voltage-gated sodium channel density and propagation velocity. The sequence of eAPs among coactive electrodes "fingerprints" neurons giving rise to these events and identifies them within neuronal ensembles. We used this property and the noninvasive nature of extracellular recording to monitor changes in excitability at multiple points in single axonal arbors simultaneously over several hours, demonstrating independence of axonal segments. Over several weeks, we recorded changes in interelectrode propagation latencies and ongoing changes in excitability in different regions of single axonal arbors. Our work illustrates how repeated eAP co-occurrences can be used to extract physiological data from single axons with low-density MEAs. However, repeated eAP co-occurrences lead to oversampling spikes from single neurons and thus can confound traditional spike-train analysis. NEW & NOTEWORTHY We studied action potential propagation in single axons using low-density multielectrode arrays. We unambiguously identified the neuronal sources of propagating action potentials and recorded extracellular action potentials from several positions within single axonal arbors. We found a surprisingly high density of axonal voltage-gated sodium channels responsible for a high propagation safety factor. Our experiments also demonstrate that excitability in different segments of single axons is regulated independently on timescales from hours to weeks.

Haploinsufficiency of BAZ1B contributes to Williams syndrome through transcriptional dysregulation of neurodevelopmental pathways.

Williams syndrome (WS) is a neurodevelopmental disorder caused by a genomic deletion of ∼28 genes that results in a cognitive and behavioral profile marked by overall intellectual impairment with relative strength in expressive language and hypersocial behavior. Advancements in protocols for neuron differentiation from induced pluripotent stem cells allowed us to elucidate the molecular circuitry underpinning the ontogeny of WS. In patient-derived stem cells and neurons, we determined the expression profile of the Williams-Beuren syndrome critical region-deleted genes and the genome-wide transcriptional consequences of the hemizygous genomic microdeletion at chromosome 7q11.23. Derived neurons displayed disease-relevant hallmarks and indicated novel aberrant pathways in WS neurons including over-activated Wnt signaling accompanying an incomplete neurogenic commitment. We show that haploinsufficiency of the ATP-dependent chromatin remodeler, BAZ1B, which is deleted in WS, significantly contributes to this differentiation defect. Chromatin-immunoprecipitation (ChIP-seq) revealed BAZ1B target gene functions are enriched for neurogenesis, neuron differentiation and disease-relevant phenotypes. BAZ1B haploinsufficiency caused widespread gene expression changes in neural progenitor cells, and together with BAZ1B ChIP-seq target genes, explained 42% of the transcriptional dysregulation in WS neurons. BAZ1B contributes to regulating the balance between neural precursor self-renewal and differentiation and the differentiation defect caused by BAZ1B haploinsufficiency can be rescued by mitigating over-active Wnt signaling in neural stem cells. Altogether, these results reveal a pivotal role for BAZ1B in neurodevelopment and implicate its haploinsufficiency as a likely contributor to the neurological phenotypes in WS.