Molecular landscapes of human hippocampal immature neurons across lifespan.

Immature dentate granule cells (imGCs) arising from adult hippocampal neurogenesis contribute to plasticity and unique brain functions in rodents1,2 and are dysregulated in multiple human neurological disorders3-5. Little is known about the molecular characteristics of adult human hippocampal imGCs, and even their existence is under debate1,6-8. Here we performed single-nucleus RNA sequencing aided by a validated machine learning-based analytic approach to identify imGCs and quantify their abundance in the human hippocampus at different stages across the lifespan. We identified common molecular hallmarks of human imGCs across the lifespan and observed age-dependent transcriptional dynamics in human imGCs that suggest changes in cellular functionality, niche interactions and disease relevance, that differ from those in mice9. We also found a decreased number of imGCs with altered gene expression in Alzheimer's disease. Finally, we demonstrated the capacity for neurogenesis in the adult human hippocampus with the presence of rare dentate granule cell fate-specific proliferating neural progenitors and with cultured surgical specimens. Together, our findings suggest the presence of a substantial number of imGCs in the adult human hippocampus via low-frequency de novo generation and protracted maturation, and our study reveals their molecular properties across the lifespan and in Alzheimer's disease.

Cervical lymph nodes and ovarian teratomas as germinal centres in NMDA receptor-antibody encephalitis.

Autoantibodies against the extracellular domain of the N-methyl-d-aspartate receptor (NMDAR) NR1 subunit cause a severe and common form of encephalitis. To better understand their generation, we aimed to characterize and identify human germinal centres actively participating in NMDAR-specific autoimmunization by sampling patient blood, CSF, ovarian teratoma tissue and, directly from the putative site of human CNS lymphatic drainage, cervical lymph nodes. From serum, both NR1-IgA and NR1-IgM were detected more frequently in NMDAR-antibody encephalitis patients versus controls (both P < 0.0001). Within patients, ovarian teratoma status was associated with a higher frequency of NR1-IgA positivity in serum (OR = 3.1; P < 0.0001) and CSF (OR = 3.8, P = 0.047), particularly early in disease and before ovarian teratoma resection. Consistent with this immunoglobulin class bias, ovarian teratoma samples showed intratumoral production of both NR1-IgG and NR1-IgA and, by single cell RNA sequencing, contained expanded highly-mutated IgA clones with an ovarian teratoma-restricted B cell population. Multiplex histology suggested tertiary lymphoid architectures in ovarian teratomas with dense B cell foci expressing the germinal centre marker BCL6, CD21+ follicular dendritic cells, and the NR1 subunit, alongside lymphatic vessels and high endothelial vasculature. Cultured teratoma explants and dissociated intratumoral B cells secreted NR1-IgGs in culture. Hence, ovarian teratomas showed structural and functional evidence of NR1-specific germinal centres. On exploring classical secondary lymphoid organs, B cells cultured from cervical lymph nodes of patients with NMDAR-antibody encephalitis produced NR1-IgG in 3/7 cultures, from patients with the highest serum NR1-IgG levels (P < 0.05). By contrast, NR1-IgG secretion was observed neither from cervical lymph nodes in disease controls nor in patients with adequately resected ovarian teratomas. Our multimodal evaluations provide convergent anatomical and functional evidence of NMDAR-autoantibody production from active germinal centres within both intratumoral tertiary lymphoid structures and traditional secondary lymphoid organs, the cervical lymph nodes. Furthermore, we develop a cervical lymph node sampling protocol that can be used to directly explore immune activity in health and disease at this emerging neuroimmune interface.

Amyloid-beta is present in human lymph nodes and greatly enriched in those of the cervical region.

Degradation and clearance of amyloid beta (Aß) peptide are likely critical for brain health. Animal studies have demonstrated the role of the glial-lymphatic (glymphatic) system in the clearance of Aß and other brain metabolites, but no such information has been available in humans. Here we ask whether this system contributes to the clearance of Aß from the human brain. In the absence of an applicable imaging method, we examined cervical and inguinal lymph nodes resected for cancer therapy or staging using immunohistochemistry. Aß-labeled cells were present in lymph nodes, and cervical lymph nodes showed labeled cells in far greater abundance than did inguinal nodes. This observation supports the hypothesis that the glymphatic system contributes to the clearance of Aß from the human brain.

Interactive web-based identification and visualization of transcript shared sequences.

We have developed TraC (Transcript Consensus), a web-based tool for detecting and visualizing shared sequences among two or more mRNA transcripts such as splice variants. Results including exon-exon boundaries are returned in a highly intuitive, data-rich, interactive plot that permits users to explore the similarities and differences of multiple transcript sequences. The online tool (http://labs.pathology.jhu.edu/nauen/trac/) is free to use. The source code is freely available for download (https://github.com/nauenlab/TraC).

Probing region-specific microstructure of human cortical areas using high angular and spatial resolution diffusion MRI.

Regional heterogeneity in cortical cyto- and myeloarchitecture forms the structural basis of mapping of cortical areas in the human brain. In this study, we investigate the potential of diffusion MRI to probe the microstructure of cortical gray matter and its region-specific heterogeneity across cortical areas in the fixed human brain. High angular resolution diffusion imaging (HARDI) data at an isotropic resolution of 92-µm and 30 diffusion-encoding directions were acquired using a 3D diffusion-weighted gradient-and-spin-echo sequence, from prefrontal (Brodmann area 9), primary motor (area 4), primary somatosensory (area 3b), and primary visual (area 17) cortical specimens (n=3 each) from three human subjects. Further, the diffusion MR findings in these cortical areas were compared with histological silver impregnation of the same specimens, in order to investigate the underlying architectonic features that constitute the microstructural basis of diffusion-driven contrasts in cortical gray matter. Our data reveal distinct and region-specific diffusion MR contrasts across the studied areas, allowing delineation of intracortical bands of tangential fibers in specific layers-layer I, layer VI, and the inner and outer bands of Baillarger. The findings of this work demonstrate unique sensitivity of diffusion MRI to differentiate region-specific cortical microstructure in the human brain, and will be useful for myeloarchitectonic mapping of cortical areas as well as to achieve an understanding of the basis of diffusion NMR contrasts in cortical gray matter.

Lymphocyte-Activation Gene 3 Facilitates Pathological Tau Neuron-to-Neuron Transmission.

The spread of prion-like protein aggregates is a common driver of pathogenesis in various neurodegenerative diseases, including Alzheimer's disease (AD) and related Tauopathies. Tau pathologies exhibit a clear progressive spreading pattern that correlates with disease severity. Clinical observation combined with complementary experimental studies has shown that Tau preformed fibrils (PFF) are prion-like seeds that propagate pathology by entering cells and templating misfolding and aggregation of endogenous Tau. While several cell surface receptors of Tau are known, they are not specific to the fibrillar form of Tau. Moreover, the underlying cellular mechanisms of Tau PFF spreading remain poorly understood. Here, it is shown that the lymphocyte-activation gene 3 (Lag3) is a cell surface receptor that binds to PFF but not the monomer of Tau. Deletion of Lag3 or inhibition of Lag3 in primary cortical neurons significantly reduces the internalization of Tau PFF and subsequent Tau propagation and neuron-to-neuron transmission. Propagation of Tau pathology and behavioral deficits induced by injection of Tau PFF in the hippocampus and overlying cortex are attenuated in mice lacking Lag3 selectively in neurons. These results identify neuronal Lag3 as a receptor of pathologic Tau in the brain，and for AD and related Tauopathies, a therapeutic target.

Deep learning-based optical coherence tomography image analysis of human brain cancer.

Real-time intraoperative delineation of cancer and non-cancer brain tissues, especially in the eloquent cortex, is critical for thorough cancer resection, lengthening survival, and improving quality of life. Prior studies have established that thresholding optical attenuation values reveals cancer regions with high sensitivity and specificity. However, threshold of a single value disregards local information important to making more robust predictions. Hence, we propose deep convolutional neural networks (CNNs) trained on labeled OCT images and co-occurrence matrix features extracted from these images to synergize attenuation characteristics and texture features. Specifically, we adapt a deep ensemble model trained on 5,831 examples in a training dataset of 7 patients. We obtain 93.31% sensitivity and 97.04% specificity on a holdout set of 4 patients without the need for beam profile normalization using a reference phantom. The segmentation maps produced by parsing the OCT volume and tiling the outputs of our model are in excellent agreement with attenuation mapping-based methods. Our new approach for this important application has considerable implications for clinical translation.

Pathological Tau transmission initiated by binding lymphocyte-activation gene 3.

The spread of prion-like protein aggregates is believed to be a common driver of pathogenesis in many neurodegenerative diseases. Accumulated tangles of filamentous Tau protein are considered pathogenic lesions of Alzheimer's disease (AD) and related Tauopathies, including progressive supranuclear palsy, and corticobasal degeneration. Tau pathologies in these illnesses exhibits a clear progressive and hierarchical spreading pattern that correlates with disease severity1,2. Clinical observation combined with complementary experimental studies3,4 have shown that Tau preformed fibrils (PFF) are prion-like seeds that propagate pathology by entering cells and templating misfolding and aggregation of endogenous Tau. While several receptors of Tau are known, they are not specific to the fibrillar form of Tau. Moreover, the underlying cellular mechanisms of Tau PFF spreading remains poorly understood. Here, we show that the lymphocyte-activation gene 3 (Lag3) is a cell surface receptor that binds to PFF, but not monomer, of Tau. Deletion of Lag3 or inhibition of Lag3 in primary cortical neurons significantly reduces the internalization of Tau PFF and subsequent Tau propagation and neuron-to-neuron transmission. Propagation of Tau pathology and behavioral deficits induced by injection of Tau PFF in the hippocampus and overlying cortex are attenuated in mice lacking Lag3 selectively in neurons. Our results identify neuronal Lag3 as a receptor of pathologic Tau in the brain, and for AD and related Tauopathies a therapeutic target.

A single-cell transcriptome atlas of glial diversity in the human hippocampus across the postnatal lifespan.

The molecular diversity of glia in the human hippocampus and their temporal dynamics over the lifespan remain largely unknown. Here, we performed single-nucleus RNA sequencing to generate a transcriptome atlas of the human hippocampus across the postnatal lifespan. Detailed analyses of astrocytes, oligodendrocyte lineages, and microglia identified subpopulations with distinct molecular signatures and revealed their association with specific physiological functions, age-dependent changes in abundance, and disease relevance. We further characterized spatiotemporal heterogeneity of GFAP-enriched astrocyte subpopulations in the hippocampal formation using immunohistology. Leveraging glial subpopulation classifications as a reference map, we revealed the diversity of glia differentiated from human pluripotent stem cells and identified dysregulated genes and pathological processes in specific glial subpopulations in Alzheimer's disease (AD). Together, our study significantly extends our understanding of human glial diversity, population dynamics across the postnatal lifespan, and dysregulation in AD and provides a reference atlas for stem-cell-based glial differentiation.

Rapidly fatal infection with Bacillus cereus/thuringiensis: genome assembly of the responsible pathogen and consideration of possibly contributing toxins.

Bloodstream infection with Bacillus cereus/thuringiensis can be life threatening, particularly in patients who are severely immunocompromised. In this report we describe a case that progressed from asymptomatic to fatal over approximately 5 hours despite extensive resuscitation efforts. We identify the pathogen and assemble its genome, in which we find genes for toxins that may have contributed to the precipitous demise. In the context of this and other cases we discuss the possible indication for rapid appropriate antibiotic administration and potentially antitoxin treatment or toxin removal in fulminant illness in immunocompromised patients.

Label-free imaging of human brain tissue at subcellular resolution for potential rapid intra-operative assessment of glioma surgery.

Background: Frozen section and smear preparation are the current standard for intraoperative histopathology during cancer surgery. However, these methods are time-consuming and subject to limited sampling. Multiphoton microscopy (MPM) is a high-resolution non-destructive imaging technique capable of optical sectioning in real time with subcellular resolution. In this report, we systematically investigated the feasibility and translation potential of MPM for rapid histopathological assessment of label- and processing-free surgical specimens. Methods: We employed a customized MPM platform to capture architectural and cytological features of biological tissues based on two-photon excited NADH and FAD autofluorescence and second harmonic generation from collagen. Infiltrating glioma, an aggressive disease that requires subcellular resolution for definitive characterization during surgery, was chosen as an example for this validation study. MPM images were collected from resected brain specimens of 19 patients and correlated with histopathology. Deep learning was introduced to assist with image feature recognition. Results: MPM robustly captures diagnostic features of glioma including increased cellularity, cellular and nuclear pleomorphism, microvascular proliferation, necrosis, and collagen deposition. Preliminary application of deep learning to MPM images achieves high accuracy in distinguishing gray from white matter and cancer from non-cancer. We also demonstrate the ability to obtain such images from intact brain tissue with a multiphoton endomicroscope for intraoperative application. Conclusion: Multiphoton imaging correlates well with histopathology and is a promising tool for characterization of cancer and delineation of infiltration within seconds during brain surgery.

A case of luteinizing thecoma with sclerosing peritonitis: revisiting a link with anti-epileptic drugs.

Luteinizing thecoma with sclerosing peritonitis (LTSP) is a rare ovarian tumor of unclear etiology and pathogenesis. The diagnostic entity was proposed in 1994, but a number of earlier reports described possible cases, and some suggested an association with anti-epileptic drugs (AEDs). In presenting a new case we review the literature of previous cases to evaluate the possibility of such a link. When cases in reproductively immature patients are considered, evidence for an association between LTSP and AEDs is strongly suggested despite the rarity of the condition.

Putative Autoantigen Leiomodin-1 Is Expressed in the Human Brain and in the Membrane Fraction of Newly Formed Neurons.

Nodding syndrome is a pediatric epilepsy disorder associated with Onchocerca volvulus infection, but the mechanism driving this relationship is unclear. One hypothesis proposes that parasite-induced immune responses cross-react with human leiomodin-1 resulting in immune-mediated central nervous system (CNS) damage. However, as leiomodin-1 expression and epitope availability in human neurons remains uncharacterized, the relevance of leiomodin-1 autoimmunity is unknown. Leiomodin-1 transcript expression was assessed in silico using publicly available ribonucleic acid (RNA) sequencing databases and in tissue by in situ hybridization and quantitative polymerase chain reaction. Abundance and subcellular localization were examined by cell fractionation and immunoblotting. Leiomodin-1 transcripts were expressed in cells of the CNS, including neurons and astrocytes. Protein was detectable from all brain regions examined as well as from representative cell lines and in vitro differentiated neurons and astrocytes. Leiomodin-1 was expressed on the membrane of newly formed neurons, but not neural progenitor cells or mature neurons. Importantly, leiomodin-1 antibodies were only toxic to cells expressing leiomodin-1 on the membrane. Our findings provide evidence that leiomodin-1 is expressed in human neurons and glia. Furthermore, we show membrane expression mediates leiomodin-1 antibody toxicity, suggesting these antibodies may play a role in pathogenesis.

Coactivation and timing-dependent integration of synaptic potentiation and depression.

Neuronal synaptic connections can be potentiated or depressed by paired pre- and postsynaptic spikes, depending on the spike timing. We show that in cultured rat hippocampal neurons a calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated potentiation process and a calcineurin-mediated depression process can be activated concomitantly by spike triplets or quadruplets. The integration of the two processes critically depends on their activation timing. Depression can cancel previously activated potentiation, whereas potentiation tends to override previously activated depression. The time window for potentiation to dominate is about 70 ms, beyond which the two processes cancel. These results indicate that the signaling machinery underlying spike timing-dependent plasticity (STDP) may be separated into functional modules that are sensitive to the spatiotemporal dynamics (rather than the amount) of calcium influx. The timing dependence of modular interaction provides a quantitative framework for understanding the temporal integration of STDP.

Extra-central nervous system target for assessment and treatment in refractory anti-N-methyl-d-aspartate receptor encephalitis.

Anti-N-methyl-d-aspartate-type glutamate receptor autoimmune encephalitis can arise in the setting of ovarian teratoma and often responds to resection. When it occurs in the absence of tumor, failure to respond to treatment may be more likely, and affected patients often require intensive care. To further understand the mechanisms and potential management, we present findings from an autopsy conducted on a young woman who died of refractory autoimmune encephalitis of this type. Rituximab was administered 70 days before death, and both 37 and 14 days before death, CD19+ lymphocytes were only 0.1% of blood cells. Ten sessions of plasmapheresis were performed after rituximab treatment. Nonetheless, the autoantibodies were present in serum 4 days before death, demonstrating ongoing antibody production. The hippocampus and medial temporal lobe demonstrated inflammation with T cell and prominent microglial involvement, but no plasma cells or plasmablasts were found there, or anywhere in the brain, despite an extensive search. Examination of lymph node tissue identified many plasma cells along sinusoids. These findings demonstrate that the antibody-producing cells are long-lived and can reside in lymphoid tissue. Awareness of continuing antibody production, the extra-central nervous system site, the indication for cytotoxic therapy, and the potential for biopsy assessment may lead to more effective treatment.

Chromosomal defects track tumor subpopulations and change in progression in oligodendroglioma.

To assess karyotypic changes and tumor subpopulations in progression of oligodendroglioma (ODG) we analyzed histologically diagnosed 1p/19q codeleted cases using single nucleotide polymorphism (SNP) microarray data. We separated cases according to grade, which was assigned blind to karyotype information beyond 1p/19q status. The 51 WHO grade II (O2) and 18 WHO grade III (O3) specimens showed frequent chromosomal locations and patterns of change including loss of heterozygosity (LOH), often copy-neutral, on 9p and LOH on 4p and 4q together. Analysis of co-occurrence indicated that most defects were independent but also suggested increased likelihood of defects on 11q, 13q, and 14q in the presence of defects on 18, 4, and 9, respectively. We used the relative degree of change in B-allele frequency as an indicator of an abnormality's extent, and we present simulated data to clarify how information on subpopulations was thus inferred. Among 9p defects, 89.3% involved the whole tumor, whereas only 47.6% of 4q defects did so. We modeled extent through the tumor as due to a karyotypic change's likelihood of occurring and the fitness it confers on its subpopulation, and used group data to estimate these values. To assess progression directly, we evaluated specimens from six patients who underwent multiple resections since 1996. Four of these patients had received no chemotherapy or radiation, permitting assessment of the natural history of the tumor karyotype in situ. Defects present throughout a tumor at first resection remained so, whereas among subpopulations, some expanded, some remained constant, and some disappeared. The rate of expansion among subpopulations that did so was not uniform, and estimates of fitness predicted subpopulation composition at recurrence. These results extend prior studies of increased karyotypic abnormality in progression of oligodendroglioma and reveal the complex dynamics of subpopulations in the tumor over time.

Single-Cell RNA-Seq with Waterfall Reveals Molecular Cascades underlying Adult Neurogenesis.

Somatic stem cells contribute to tissue ontogenesis, homeostasis, and regeneration through sequential processes. Systematic molecular analysis of stem cell behavior is challenging because classic approaches cannot resolve cellular heterogeneity or capture developmental dynamics. Here we provide a comprehensive resource of single-cell transcriptomes of adult hippocampal quiescent neural stem cells (qNSCs) and their immediate progeny. We further developed Waterfall, a bioinformatic pipeline, to statistically quantify singe-cell gene expression along a de novo reconstructed continuous developmental trajectory. Our study reveals molecular signatures of adult qNSCs, characterized by active niche signaling integration and low protein translation capacity. Our analyses further delineate molecular cascades underlying qNSC activation and neurogenesis initiation, exemplified by decreased extrinsic signaling capacity, primed translational machinery, and regulatory switches in transcription factors, metabolism, and energy sources. Our study reveals the molecular continuum underlying adult neurogenesis and illustrates how Waterfall can be used for single-cell omics analyses of various continuous biological processes.