Enhancing mitosis quantification and detection in meningiomas with computational digital pathology.

Mitosis is a critical criterion for meningioma grading. However, pathologists' assessment of mitoses is subject to significant inter-observer variation due to challenges in locating mitosis hotspots and accurately detecting mitotic figures. To address this issue, we leverage digital pathology and propose a computational strategy to enhance pathologists' mitosis assessment. The strategy has two components: (1) A depth-first search algorithm that quantifies the mathematically maximum mitotic count in 10 consecutive high-power fields, which can enhance the preciseness, especially in cases with borderline mitotic count. (2) Implementing a collaborative sphere to group a set of pathologists to detect mitoses under each high-power field, which can mitigate subjective random errors in mitosis detection originating from individual detection errors. By depth-first search algorithm (1) , we analyzed 19 meningioma slides and discovered that the proposed algorithm upgraded two borderline cases verified at consensus conferences. This improvement is attributed to the algorithm's ability to quantify the mitotic count more comprehensively compared to other conventional methods of counting mitoses. In implementing a collaborative sphere (2) , we evaluated the correctness of mitosis detection from grouped pathologists and/or pathology residents, where each member of the group annotated a set of 48 high-power field images for mitotic figures independently. We report that groups with sizes of three can achieve an average precision of 0.897 and sensitivity of 0.699 in mitosis detection, which is higher than an average pathologist in this study (precision: 0.750, sensitivity: 0.667). The proposed computational strategy can be integrated with artificial intelligence workflow, which envisions the future of achieving a rapid and robust mitosis assessment by interactive assisting algorithms that can ultimately benefit patient management.

Nuclear RNA catabolism controls endogenous retroviruses, gene expression asymmetry, and dedifferentiation.

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.

Lifelong restructuring of 3D genome architecture in cerebellar granule cells.

The cerebellum contains most of the neurons in the human brain and exhibits distinctive modes of development and aging. In this work, by developing our single-cell three-dimensional (3D) genome assay-diploid chromosome conformation capture, or Dip-C-into population-scale (Pop-C) and virus-enriched (vDip-C) modes, we resolved the first 3D genome structures of single cerebellar cells, created life-spanning 3D genome atlases for both humans and mice, and jointly measured transcriptome and chromatin accessibility during development. We found that although the transcriptome and chromatin accessibility of cerebellar granule neurons mature in early postnatal life, 3D genome architecture gradually remodels throughout life, establishing ultra-long-range intrachromosomal contacts and specific interchromosomal contacts that are rarely seen in neurons. These results reveal unexpected evolutionarily conserved molecular processes that underlie distinctive features of neural development and aging across the mammalian life span.

Cerebellar Granule Cells Develop Non-neuronal 3D Genome Architecture over the Lifespan.

The cerebellum contains most of the neurons in the human brain, and exhibits unique modes of development, malformation, and aging. For example, granule cells-the most abundant neuron type-develop unusually late and exhibit unique nuclear morphology. Here, by developing our high-resolution single-cell 3D genome assay Dip-C into population-scale (Pop-C) and virus-enriched (vDip-C) modes, we were able to resolve the first 3D genome structures of single cerebellar cells, create life-spanning 3D genome atlases for both human and mouse, and jointly measure transcriptome and chromatin accessibility during development. We found that while the transcriptome and chromatin accessibility of human granule cells exhibit a characteristic maturation pattern within the first year of postnatal life, 3D genome architecture gradually remodels throughout life into a non-neuronal state with ultra-long-range intra-chromosomal contacts and specific inter-chromosomal contacts. This 3D genome remodeling is conserved in mice, and robust to heterozygous deletion of chromatin remodeling disease-associated genes (Chd8 or Arid1b). Together these results reveal unexpected and evolutionarily-conserved molecular processes underlying the unique development and aging of the mammalian cerebellum.

Molecular signatures underlying neurofibrillary tangle susceptibility in Alzheimer's disease.

Tau aggregation in neurofibrillary tangles (NFTs) is closely associated with neurodegeneration and cognitive decline in Alzheimer's disease (AD). However, the molecular signatures that distinguish between aggregation-prone and aggregation-resistant cell states are unknown. We developed methods for the high-throughput isolation and transcriptome profiling of single somas with NFTs from the human AD brain, quantified the susceptibility of 20 neocortical subtypes for NFT formation and death, and identified both shared and cell-type-specific signatures. NFT-bearing neurons shared a marked upregulation of synaptic transmission-related genes, including a core set of 63 genes enriched for synaptic vesicle cycling. Oxidative phosphorylation and mitochondrial dysfunction were highly cell-type dependent. Apoptosis was only modestly enriched, and the susceptibilities of NFT-bearing and NFT-free neurons for death were highly similar. Our analysis suggests that NFTs represent cell-type-specific responses to stress and synaptic dysfunction. We provide a resource for biomarker discovery and the investigation of tau-dependent and tau-independent mechanisms of neurodegeneration.

Divergent Cortical Tau Positron Emission Tomography Patterns Among Patients With Preclinical Alzheimer Disease.

Importance: Characterization of early tau deposition in individuals with preclinical Alzheimer disease (AD) is critical for prevention trials that aim to select individuals at risk for AD and halt the progression of disease. Objective: To evaluate the prevalence of cortical tau positron emission tomography (PET) heterogeneity in a large cohort of clinically unimpaired older adults with elevated ß-amyloid (A+). Design, Setting, and Participants: This cross-sectional study examined prerandomized tau PET, amyloid PET, structural magnetic resonance imaging, demographic, and cognitive data from the Anti-Amyloid Treatment in Asymptomatic AD (A4) Study from April 2014 to December 2017. Follow-up analyses used observational tau PET data from the Alzheimer's Disease Neuroimaging Initiative (ADNI), the Harvard Aging Brain Study (HABS), and the Wisconsin Registry for Alzheimer's Prevention and the Wisconsin Alzheimer's Disease Research Center (together hereinafter referred to as Wisconsin) to evaluate consistency. Participants were clinically unimpaired at the study visit closest to the tau PET scan and had available amyloid and tau PET data (A4 Study, n = 447; ADNI, n = 433; HABS, n = 190; and Wisconsin, n = 328). No participants who met eligibility criteria were excluded. Data were analyzed from May 11, 2021, to January 25, 2022. Main Outcomes and Measures: Individuals with preclinical AD with heterogeneous cortical tau PET patterns (A+T cortical+) were identified by examining asymmetrical cortical tau signal and disproportionate cortical tau signal relative to medial temporal lobe (MTL) tau. Voxelwise tau patterns, amyloid, neurodegeneration, cognition, and demographic characteristics were examined. Results: The 447 A4 participants (A+ group, 392; and normal ß-amyloid group, 55), with a mean (SD) age of 71.8 (4.8) years, included 239 women (54%). A total of 36 individuals in the A+ group (9% of the A+ group) exhibited heterogeneous cortical tau patterns and were further categorized into 3 subtypes: asymmetrical left, precuneus dominant, and asymmetrical right. A total of 116 individuals in the A+ group (30% of the A+ group) showed elevated MTL tau (A+T MTL+). Individuals in the A+T cortical+ group were younger than those in the A+T MTL+ group (t61.867 = -2.597; P = .03). Across the A+T cortical+ and A+T MTL+ groups, increased regional tau was associated with reduced hippocampal volume and MTL thickness but not with cortical thickness. Memory scores were comparable between the A+T cortical+ and A+T MTL+ groups, whereas executive functioning scores were lower for the A+T cortical+ group than for the A+T MTL+ group. The prevalence of the A+T cortical+ group and tau patterns within the A+T cortical+ group were consistent in ADNI, HABS, and Wisconsin. Conclusions and Relevance: This study suggests that early tau deposition may follow multiple trajectories during preclinical AD and may involve several cortical regions. Staging procedures, especially those based on neuropathology, that assume a uniform trajectory across individuals are insufficient for disease monitoring with tau imaging.

Whole-Transcriptome Profiling and circRNA-miRNA-mRNA Regulatory Networks in B-Cell Development.

The generation and differentiation of B lymphocytes (B cells) is a flexible process with many critical regulatory factors. Previous studies indicated that non-coding RNAs play multiple roles in the development of lymphocytes. However, little has been known about the circular RNA (circRNA) profiles and their competing endogenous RNA (ceRNA) networks in B-cell development and differentiation. Here, four B-cell subsets were purified from single-cell suspensions of mouse bone marrow. Then RNA sequencing (RNA-Seq) was used to display expression profiles of circRNAs, miRNAs and mRNAs during B-cell differentiation. 175, 203, 219 and 207 circRNAs were specifically expressed in pro-B cells, pre-B cells, immature B cells and mature B cells, respectively. The circRNA-associated ceRNA networks constructed in two sequential stages of B-cell differentiation revealed the potential mechanism of circRNAs in these processes. This study is the first to explore circRNA profiles and circRNA-miRNA-mRNA networks in different B-cell developmental stages of mouse bone marrow, which contribute to further research on their mechanism in B-cell development and differentiation.

Human Astrocytes Exhibit Tumor Microenvironment-, Age-, and Sex-Related Transcriptomic Signatures.

Astrocytes are critical for the development and function of synapses. There are notable species differences between human astrocytes and commonly used animal models. Yet, it is unclear whether astrocytic genes involved in synaptic function are stable or exhibit dynamic changes associated with disease states and age in humans, which is a barrier in understanding human astrocyte biology and its potential involvement in neurologic diseases. To better understand the properties of human astrocytes, we acutely purified astrocytes from the cerebral cortices of over 40 humans across various ages, sexes, and disease states. We performed RNA sequencing to generate transcriptomic profiles of these astrocytes and identified genes associated with these biological variables. We found that human astrocytes in tumor-surrounding regions downregulate genes involved in synaptic function and sensing of signals in the microenvironment, suggesting involvement of peritumor astrocytes in tumor-associated neural circuit dysfunction. In aging, we also found downregulation of synaptic regulators and upregulation of markers of cytokine signaling, while in maturation we identified changes in ionic transport with implications for calcium signaling. In addition, we identified subtle sexual dimorphism in human cortical astrocytes, which has implications for observed sex differences across many neurologic disorders. Overall, genes involved in synaptic function exhibit dynamic changes in the peritumor microenvironment and aging. These data provide powerful new insights into human astrocyte biology in several biologically relevant states that will aid in generating novel testable hypotheses about homeostatic and reactive astrocytes in humans.SIGNIFICANCE STATEMENT Astrocytes are an abundant class of cells playing integral roles at synapses. Astrocyte dysfunction is implicated in a variety of human neurologic diseases. Yet our knowledge of astrocytes is largely based on mouse studies. Direct knowledge of human astrocyte biology remains limited. Here, we present transcriptomic profiles of human cortical astrocytes, and we identified molecular differences associated with age, sex, and disease state. We found that peritumor and aging astrocytes downregulate genes involved in astrocyte-synapse interactions. These data provide necessary insight into human astrocyte biology that will improve our understanding of human disease.

The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C.

Lysosome dysfunction is a shared feature of rare lysosomal storage diseases and common age-related neurodegenerative diseases. Microglia, the brain-resident macrophages, are particularly vulnerable to lysosome dysfunction because of the phagocytic stress of clearing dying neurons, myelin, and debris. CD22 is a negative regulator of microglial homeostasis in the aging mouse brain, and soluble CD22 (sCD22) is increased in the cerebrospinal fluid of patients with Niemann-Pick type C disease (NPC). However, the role of CD22 in the human brain remains unknown. In contrast to previous findings in mice, here, we show that CD22 is expressed by oligodendrocytes in the human brain and binds to sialic acid­dependent ligands on microglia. Using unbiased genetic and proteomic screens, we identify insulin-like growth factor 2 receptor (IGF2R) as the binding partner of sCD22 on human myeloid cells. Targeted truncation of IGF2R revealed that sCD22 docks near critical mannose 6-phosphate­binding domains, where it disrupts lysosomal protein trafficking. Interfering with the sCD22-IGF2R interaction using CD22 blocking antibodies ameliorated lysosome dysfunction in human NPC1 mutant induced pluripotent stem cell­derived microglia-like cells without harming oligodendrocytes in vitro. These findings reinforce the differences between mouse and human microglia and provide a candidate microglia-directed immunotherapeutic to treat NPC.

Defining the nature of human pluripotent stem cell-derived interneurons via single-cell analysis.

The specification of inhibitory neurons has been described for the mouse and human brain, and many studies have shown that pluripotent stem cells (PSCs) can be used to create interneurons in vitro. It is unclear whether in vitro methods to produce human interneurons generate all the subtypes found in brain, and how similar in vitro and in vivo interneurons are. We applied single-nuclei and single-cell transcriptomics to model interneuron development from human cortex and interneurons derived from PSCs. We provide a direct comparison of various in vitro interneuron derivation methods to determine the homogeneity achieved. We find that PSC-derived interneurons capture stages of development prior to mid-gestation, and represent a minority of potential subtypes found in brain. Comparison with those found in fetal or adult brain highlighted decreased expression of synapse-related genes. These analyses highlight the potential to tailor the method of generation to drive formation of particular subtypes.

Dysregulation of brain and choroid plexus cell types in severe COVID-19.

Although SARS-CoV-2 primarily targets the respiratory system, patients with and survivors of COVID-19 can suffer neurological symptoms1-3. However, an unbiased understanding of the cellular and molecular processes that are affected in the brains of patients with COVID-19 is missing. Here we profile 65,309 single-nucleus transcriptomes from 30 frontal cortex and choroid plexus samples across 14 control individuals (including 1 patient with terminal influenza) and 8 patients with COVID-19. Although our systematic analysis yields no molecular traces of SARS-CoV-2 in the brain, we observe broad cellular perturbations indicating that barrier cells of the choroid plexus sense and relay peripheral inflammation into the brain and show that peripheral T cells infiltrate the parenchyma. We discover microglia and astrocyte subpopulations associated with COVID-19 that share features with pathological cell states that have previously been reported in human neurodegenerative disease4-6. Synaptic signalling of upper-layer excitatory neurons-which are evolutionarily expanded in humans7 and linked to cognitive function8-is preferentially affected in COVID-19. Across cell types, perturbations associated with COVID-19 overlap with those found in chronic brain disorders and reside in genetic variants associated with cognition, schizophrenia and depression. Our findings and public dataset provide a molecular framework to understand current observations of COVID-19-related neurological disease, and any such disease that may emerge at a later date.

Hepatic arginase deficiency fosters dysmyelination during postnatal CNS development.

Deficiency of arginase is associated with hyperargininemia, and prominent features include spastic diplegia/tetraplegia, clonus, and hyperreflexia; loss of ambulation, intellectual disability and progressive neurological decline are other signs. To gain greater insight into the unique neuromotor features, we performed gene expression profiling of the motor cortex of a murine model of the disorder. Coexpression network analysis suggested an abnormality with myelination, which was supported by limited existing human data. Utilizing electron microscopy, marked dysmyelination was detected in 2-week-old homozygous Arg1-KO mice. The corticospinal tract was found to be adversely affected, supporting dysmyelination as the cause of the unique neuromotor features and implicating oligodendrocyte impairment in a deficiency of hepatic Arg1. Following neonatal hepatic gene therapy to express Arg1, the subcortical white matter, pyramidal tract, and corticospinal tract all showed a remarkable recovery in terms of myelinated axon density and ultrastructural integrity with active wrapping of axons by nearby oligodendrocyte processes. These findings support the following conclusions: arginase deficiency is a leukodystrophy affecting the brain and spinal cord while sparing the peripheral nervous system, and neonatal AAV hepatic gene therapy can rescue the defects associated with myelinated axons, strongly implicating the functional recovery of oligodendrocytes after restoration of hepatic arginase activity.

Nav1.1-Overexpressing Interneuron Transplants Restore Brain Rhythms and Cognition in a Mouse Model of Alzheimer's Disease.

Inhibitory interneurons regulate the oscillatory rhythms and network synchrony that are required for cognitive functions and disrupted in Alzheimer's disease (AD). Network dysrhythmias in AD and multiple neuropsychiatric disorders are associated with hypofunction of Nav1.1, a voltage-gated sodium channel subunit predominantly expressed in interneurons. We show that Nav1.1-overexpressing, but not wild-type, interneuron transplants derived from the embryonic medial ganglionic eminence (MGE) enhance behavior-dependent gamma oscillatory activity, reduce network hypersynchrony, and improve cognitive functions in human amyloid precursor protein (hAPP)-transgenic mice, which simulate key aspects of AD. Increased Nav1.1 levels accelerated action potential kinetics of transplanted fast-spiking and non-fast-spiking interneurons. Nav1.1-deficient interneuron transplants were sufficient to cause behavioral abnormalities in wild-type mice. We conclude that the efficacy of interneuron transplantation and the function of transplanted cells in an AD-relevant context depend on their Nav1.1 levels. Disease-specific molecular optimization of cell transplants may be required to ensure therapeutic benefits in different conditions.

Dlx1 and Dlx2 Promote Interneuron GABA Synthesis, Synaptogenesis, and Dendritogenesis.

The postnatal functions of the Dlx1&2 transcription factors in cortical interneurons (CINs) are unknown. Here, using conditional Dlx1, Dlx2, and Dlx1&2 knockouts (CKOs), we defined their roles in specific CINs. The CKOs had dendritic, synaptic, and survival defects, affecting even PV+ CINs. We provide evidence that DLX2 directly drives Gad1, Gad2, and Vgat expression, and show that mutants had reduced mIPSC amplitude. In addition, the mutants formed fewer GABAergic synapses on excitatory neurons and had reduced mIPSC frequency. Furthermore, Dlx1/2 CKO had hypoplastic dendrites, fewer excitatory synapses, and reduced excitatory input. We provide evidence that some of these phenotypes were due to reduced expression of GRIN2B (a subunit of the NMDA receptor), a high confidence Autism gene. Thus, Dlx1&2 coordinate key components of CIN postnatal development by promoting their excitability, inhibitory output, and survival.

GABAergic Interneuron Differentiation in the Basal Forebrain Is Mediated through Direct Regulation of Glutamic Acid Decarboxylase Isoforms by Dlx Homeobox Transcription Factors.

GABA is the key inhibitory neurotransmitter in the cortex but regulation of its synthesis during forebrain development is poorly understood. In the telencephalon, members of the distal-less (Dlx) homeobox gene family are expressed in, and regulate the development of, the basal ganglia primodia from which many GABAergic neurons originate and migrate to other forebrain regions. The Dlx1/Dlx2 double knock-out mice die at birth with abnormal cortical development, including loss of tangential migration of GABAergic inhibitory interneurons to the neocortex (Anderson et al., 1997a). We have discovered that specific promoter regulatory elements of glutamic acid decarboxylase isoforms (Gad1 and Gad2), which regulate GABA synthesis from the excitatory neurotransmitter glutamate, are direct transcriptional targets of both DLX1 and DLX2 homeoproteins in vivo Further gain- and loss-of-function studies in vitro and in vivo demonstrated that both DLX1 and DLX2 are necessary and sufficient for Gad gene expression. DLX1 and/or DLX2 activated the transcription of both Gad genes, and defects in Dlx function disrupted the differentiation of GABAergic interneurons with global reduction in GABA levels in the forebrains of the Dlx1/Dlx2 double knock-out mouse in vivo Identification of Gad genes as direct Dlx transcriptional targets is significant; it extends our understanding of Dlx gene function in the developing forebrain beyond the regulation of tangential interneuron migration to the differentiation of GABAergic interneurons arising from the basal telencephalon, and may help to unravel the pathogenesis of several developmental brain disorders.SIGNIFICANCE STATEMENT GABA is the major inhibitory neurotransmitter in the brain. We show that Dlx1/Dlx2 homeobox genes regulate GABA synthesis during forebrain development through direct activation of glutamic acid decarboxylase enzyme isoforms that convert glutamate to GABA. This discovery helps explain how Dlx mutations result in abnormal forebrain development, due to defective differentiation, in addition to the loss of tangential migration of GABAergic inhibitory interneurons to the neocortex. Reduced numbers or function of cortical GABAergic neurons may lead to hyperactivity states such as seizures (Cobos et al., 2005) or contribute to the pathogenesis of some autism spectrum disorders. GABAergic dysfunction in the basal ganglia could disrupt the learning and development of complex motor and cognitive behaviors (Rubenstein and Merzenich, 2003).

Human von Economo neurons express transcription factors associated with Layer V subcerebral projection neurons.

The von Economo neurons (VENs) are large bipolar Layer V projection neurons found chiefly in the anterior cingulate and frontoinsular cortices. Although VENs have been linked to prevalent illnesses such as frontotemporal dementia, autism, and schizophrenia, little is known about VEN identity, including their major projection targets. Here, we undertook a developmental transcription factor expression study, focusing on markers associated with specific classes of Layer V projection neurons. Using mRNA in situ hybridization, we found that VENs prominently express FEZF2 and CTIP2, transcription factors that regulate the fate and differentiation of subcerebral projection neurons, in humans aged 3 months to 65 years. In contrast, few VENs expressed markers associated with callosal or corticothalamic projections. These findings suggest that VENs may represent a specialized Layer V projection neuron for linking cortical autonomic control sites to brainstem or spinal cord regions.

Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model.

Alzheimer's disease (AD) results in cognitive decline and altered network activity, but the mechanisms are unknown. We studied human amyloid precursor protein (hAPP) transgenic mice, which simulate key aspects of AD. Electroencephalographic recordings in hAPP mice revealed spontaneous epileptiform discharges, indicating network hypersynchrony, primarily during reduced gamma oscillatory activity. Because this oscillatory rhythm is generated by inhibitory parvalbumin (PV) cells, network dysfunction in hAPP mice might arise from impaired PV cells. Supporting this hypothesis, hAPP mice and AD patients had decreased levels of the interneuron-specific and PV cell-predominant voltage-gated sodium channel subunit Nav1.1. Restoring Nav1.1 levels in hAPP mice by Nav1.1-BAC expression increased inhibitory synaptic activity and gamma oscillations and reduced hypersynchrony, memory deficits, and premature mortality. We conclude that reduced Nav1.1 levels and PV cell dysfunction critically contribute to abnormalities in oscillatory rhythms, network synchrony, and memory in hAPP mice and possibly in AD.