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Inborn errors of TLR3-dependent type I IFN immunity in cortical neurons underlie forebrain herpes simplex virus-1 (HSV-1) encephalitis (HSE) due to uncontrolled viral growth and subsequent cell death. We report an otherwise healthy patient with HSE who was compound heterozygous for nonsense (R422*) and frameshift (P493fs9*) RIPK3 variants. Receptor-interacting protein kinase 3 (RIPK3) is a ubiquitous cytoplasmic kinase regulating cell death outcomes, including apoptosis and necroptosis. In vitro, the R422* and P493fs9* RIPK3 proteins impaired cellular apoptosis and necroptosis upon TLR3, TLR4, or TNFR1 stimulation and ZBP1/DAI-mediated necroptotic cell death after HSV-1 infection. The patient’s fibroblasts displayed no detectable RIPK3 expression. After TNFR1 or TLR3 stimulation, the patient’s cells did not undergo apoptosis or necroptosis. After HSV-1 infection, the cells supported excessive viral growth despite normal induction of antiviral IFN-β and IFN-stimulated genes (ISGs). This phenotype was, nevertheless, rescued by application of exogenous type I IFN. The patient’s human pluripotent stem cell (hPSC)–derived cortical neurons displayed impaired cell death and enhanced viral growth after HSV-1 infection, as did isogenic RIPK3-knockout hPSC-derived cortical neurons. Inherited RIPK3 deficiency therefore confers a predisposition to HSE by impairing the cell death–dependent control of HSV-1 in cortical neurons but not their production of or response to type I IFNs.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection is associated with a diverse spectrum of neurological complications during the acute and postacute stages. The pathogenesis of these complications is complex and dependent on many factors. For accurate and consistent interpretation of experimental data in this fast-growing field of research, it is essential to use terminology consistently. In this article, we outline the distinctions between neuroinvasiveness, neurotropism, and neurovirulence. Additionally, we discuss current knowledge of these distinct features underlying the pathogenesis of SARS-CoV-2-associated neurological complications. Lastly, we briefly discuss the advantages and limitations of different experimental models, and how these approaches can further be leveraged to advance the field.

Viral encephalitis is a major neglected medical problem. Host defense mechanisms against viral infection of the central nervous system (CNS) have long remained unclear. The few previous studies of CNS-specific immunity to viruses in mice in vivo and humans in vitro have focused on the contributions of circulating leukocytes, resident microglial cells and astrocytes, with neurons long considered passive victims of viral infection requiring protection from extrinsic antiviral mechanisms. The last decade has witnessed the gradual emergence of the notion that neurons also combat viruses through cell-intrinsic mechanisms. Forward genetic approaches in humans have shown that monogenic inborn errors of TLR3, IFN-α/β, or snoRNA31 immunity confer susceptibility to herpes simplex virus 1 (HSV-1) infection of the forebrain, whereas inborn errors of DBR1 underlie brainstem infections due to various viruses, including HSV-1. The study of human pluripotent stem cell (hPSC)-derived CNS-resident cells has unraveled known (i.e. TLR3-dependent IFN-α/β immunity) and new (i.e. snoRNA31-dependent or DBR1-dependent immunity) cell-intrinsic antiviral mechanisms operating in neurons. Reverse genetic approaches in mice have confirmed that some known antiviral mechanisms also operate in mouse neurons (e.g. TLR3 and IFN-α/β immunity). The search for human inborn errors of immunity (IEIs) underlying various forms of viral encephalitis, coupled with mouse models in vivo, and hPSC-based culture models of CNS and peripheral nervous system cells and organoids in vitro, should shed further light on the cell-specific and tissue-specific mechanisms of host defense against viruses in the human brain.

Placodes are embryonic structures originating from the rostral ectoderm that give rise to highly diverse organs and tissues, comprising the anterior pituitary gland, paired sense organs and cranial sensory ganglia. Their development, including the underlying gene regulatory networks and signalling pathways, have been for the most part characterised in animal models. In this Review, we describe how placode development can be recapitulated by the differentiation of human pluripotent stem cells towards placode progenitors and their derivatives, highlighting the value of this highly scalable platform as an optimal in vitro tool to study the development of human placodes, and identify human-specific mechanisms in their development, function and pathology.

Contains fulltext : 244608.pdf (Publisher’s version ) (Closed access) Aberrant inflammation in the CNS has been implicated as a major player in the pathogenesis of human neurodegenerative disease. We developed a new approach to derive microglia from human pluripotent stem cells (hPSCs) and built a defined hPSC-derived tri-culture system containing pure populations of hPSC-derived microglia, astrocytes, and neurons to dissect cellular cross-talk along the neuroinflammatory axis in vitro. We used the tri-culture system to model neuroinflammation in Alzheimer's disease with hPSCs harboring the APP(SWE)+/+ mutation and their isogenic control. We found that complement C3, a protein that is increased under inflammatory conditions and implicated in synaptic loss, is potentiated in tri-culture and further enhanced in APP(SWE)+/+ tri-cultures due to microglia initiating reciprocal signaling with astrocytes to produce excess C3. Our study defines the major cellular players contributing to increased C3 in Alzheimer's disease and presents a broadly applicable platform to study neuroinflammation in human disease.

Purpose of ReviewTo improve our clinical understanding of facial onset sensory and motor neuronopathy (FOSMN).Recent FindingsWe identified 29 new cases and 71 literature cases, resulting in a cohort of 100 patients with FOSMN. During follow-up, cognitive and behavioral changes became apparent in 8 patients, suggesting that changes within the spectrum of frontotemporal dementia (FTD) are a part of the natural history of FOSMN. Another new finding was chorea, seen in 6 cases. Despite reports of autoantibodies, there is no consistent evidence to suggest an autoimmune pathogenesis. Four of 6 autopsies had TAR DNA-binding protein (TDP) 43 pathology. Seven cases had genetic mutations associated with neurodegenerative diseases.SummaryFOSMN is a rare disease with a highly characteristic onset and pattern of disease progression involving initial sensory disturbances, followed by bulbar weakness with a cranial to caudal spread of pathology. Although not conclusive, the balance of evidence suggests that FOSMN is most likely to be a TDP-43 proteinopathy within the amyotrophic lateral sclerosis–FTD spectrum.

COVID-19 patients commonly present with neurological signs of central nervous system (CNS) and/or peripheral nervous system dysfunction. However, which neural cells are permissive to infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been controversial. Here, we show that midbrain dopamine (DA) neurons derived from human pluripotent stem cells (hPSCs) are selectively permissive to SARS-CoV-2 infection both in vitro and upon transplantation in vivo, and that SARS-CoV-2 infection triggers a DA neuron inflammatory and cellular senescence response. A high-throughput screen in hPSC-derived DA neurons identified several FDA approved drugs, including riluzole, metformin, and imatinib, that can rescue the cellular senescence phenotype and prevent SARS-CoV-2 infection. RNA-seq analysis of human ventral midbrain tissue from COVID-19 patients, using formalin-fixed paraffin-embedded autopsy samples, confirmed the induction of an inflammatory and cellular senescence signature and identified low levels of SARS-CoV-2 transcripts. Our findings demonstrate that hPSC-derived DA neurons can serve as a disease model to study neuronal susceptibility to SARS-CoV-2 and to identify candidate neuroprotective drugs for COVID-19 patients. The susceptibility of hPSC-derived DA neurons to SARS-CoV-2 and the observed inflammatory and senescence transcriptional responses suggest the need for careful, long-term monitoring of neurological problems in COVID-19 patients.

Human herpes simplex virus 1 (HSV-1) encephalitis can be caused by inborn errors of the TLR3 pathway, resulting in impairment of CNS cell-intrinsic antiviral immunity. Deficiencies of the TLR3 pathway impair cell-intrinsic immunity to vesicular stomatitis virus (VSV) and HSV-1 in fibroblasts, and to HSV-1 in cortical but not trigeminal neurons. The underlying molecular mechanism is thought to involve impaired IFN-α/β induction by the TLR3 recognition of dsRNA viral intermediates or by-products. However, we show here that human TLR3 controls constitutive levels of IFNB mRNA and secreted bioactive IFN-β protein, and thereby also controls constitutive mRNA levels for IFN-stimulated genes (ISGs) in fibroblasts. Tlr3-/- mouse embryonic fibroblasts also have lower basal ISG levels. Moreover, human TLR3 controls basal levels of IFN-β secretion and ISG mRNA in induced pluripotent stem cell-derived cortical neurons. Consistently, TLR3-deficient human fibroblasts and cortical neurons are vulnerable not only to both VSV and HSV-1, but also to several other families of viruses. The mechanism by which TLR3 restricts viral growth in human fibroblasts and cortical neurons in vitro and, by inference, by which the human CNS prevents infection by HSV-1 in vivo, is therefore based on the control of early viral infection by basal IFN-β immunity. ispartof: JOURNAL OF CLINICAL INVESTIGATION vol:131 issue:1 ispartof: location:United States status: published

Advancements in human pluripotent stem cell technology offer a unique opportunity for the neuroimmunology field to study host-virus interactions directly in disease-relevant cells of the human central nervous system (CNS). Viral encephalitis is most commonly caused by herpesviruses, arboviruses and enteroviruses targeting distinct CNS cell types and often leading to severe neurological damage with poor clinical outcomes. Furthermore, different neurotropic viruses will affect the CNS at distinct developmental stages, from early prenatal brain development to the aged brain. With the unique flexibility and scalability of human pluripotent stem cell technology, it is now possible to examine the molecular mechanisms underlying acute infection and latency, determine which CNS subpopulations are specifically infected, study temporal aspects of viral susceptibility, perform high-throughput chemical or genetic screens for viral restriction factors and explore complex cell-non-autonomous disease mechanisms. Therefore, human pluripotent stem cell technology has the potential to address key unanswered questions about antiviral immunity in the CNS, including emerging questions on the potential CNS tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

International audience; Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is typically sporadic. Inborn errors of TLR3- and DBR1-mediated central nervous system cell-intrinsic immunity can account for forebrain and brainstem HSE, respectively. We report five unrelated patients with forebrain HSE, each heterozygous for one of four rare variants of SNORA31, encoding a small nucleolar RNA of the H/ACA class that are predicted to direct the isomerization of uridine residues to pseudouridine in small nuclear RNA and ribosomal RNA. We show that CRISPR/Cas9-introduced bi- and monoallelic SNORA31 deletions render human pluripotent stem cell (hPSC)-derived cortical neurons susceptible to HSV-1. Accordingly, SNORA31-mutated patient hPSC-derived cortical neurons are susceptible to HSV-1, like those from TLR3- or STAT1-deficient patients. Exogenous interferon (IFN)-β renders SNORA31- and TLR3- but not STAT1-mutated neurons resistant to HSV-1. Finally, transcriptome analysis of SNORA31-mutated neurons revealed normal responses to TLR3 and IFN-α/β stimulation but abnormal responses to HSV-1. Human SNORA31 thus controls central nervous system neuron-intrinsic immunity to HSV-1 by a distinctive mechanism.

Autoimmunity of the peripheral and central nervous system is an important cause of disease and long-term neurological disability. Autoantibodies can target both intracellular and extracellular neuronal epitopes. Autoantibodies that target cell-surface epitopes infer pathogenicity through several distinct mechanisms, while patients often respond to immunotherapy. However, the underlying pathogenesis of these autoantibodies is yet to be fully understood. Human stem cell–based disease modeling, and the rise of induced pluripotent stem cell technology in particular, has revolutionized the fields of disease modeling and therapeutic screening for neurological disorders. These human disease models offer a unique platform in which to study autoimmunity of the nervous system. Here, we take an in-depth look at the possibilities that these models provide to study neuronal autoantibodies and their underlying pathogenesis.

OBJECTIVE: We investigated the pathogenicity of immunoglobulin M (IgM) anti-GM1 antibodies in serum from patients with multifocal motor neuropathy (MMN) using human induced pluripotent stem cell (iPSC)-derived motor neurons (MNs). METHODS: iPSCs were generated from fibroblasts and differentiated into MNs. We studied the binding of IgM to MNs, their complement-activating properties, and effects on structural integrity using fluorescence and electron microscopy. Live cell imaging was used to study effects of antibody binding on MNs in the presence and absence of complement. RESULTS: IgM antibody binding to MNs was detected using sera from MMN patients with and without detectable anti-GM1 IgM antibody titers in enzyme-linked immunosorbent assay, but not with sera from (disease) controls. Competition and depletion experiments showed that antibodies specifically bound to GM1 on iPSC-derived MNs. Binding of these antibodies disrupted calcium homeostasis by both complement-dependent and complement-independent pathways. MNs showed marked axonal damage after complement activation, and reduced antibody pathogenicity following treatment with immunoglobulin preparations. INTERPRETATION: Our data provide evidence for the pathogenicity of anti-GM1 IgM antibodies in MMN patients and link their presence to the clinical characteristics of axonal damage and immunoglobulin responsiveness. This iPSC-derived disease model will facilitate diagnosis, studies on autoantibody pathogenicity, drug development, and screening in immune-mediated neuropathies. Ann Neurol 2016;80:71-88.

SARS-CoV-2 has caused the COVID-19 pandemic. There is an urgent need for physiological models to study SARS-CoV-2 infection using human disease-relevant cells. COVID-19 pathophysiology includes respiratory failure but involves other organ systems including gut, liver, heart, and pancreas. We present an experimental platform comprised of cell and organoid derivatives from human pluripotent stem cells (hPSCs). A Spike-enabled pseudo-entry virus infects pancreatic endocrine cells, liver organoids, cardiomyocytes, and dopaminergic neurons. Recent clinical studies show a strong association with COVID-19 and diabetes. We find that human pancreatic beta cells and liver organoids are highly permissive to SARS-CoV-2 infection, further validated using adult primary human islets and adult hepatocyte and cholangiocyte organoids. SARS-CoV-2 infection caused striking expression of chemokines, as also seen in primary human COVID-19 pulmonary autopsy samples. hPSC-derived cells/organoids provide valuable models for understanding the cellular responses of human tissues to SARS-CoV-2 infection and for disease modeling of COVID-19.

Herpes simplex virus type 1 (HSV-1) encephalitis (HSE) is the most common sporadic viral encephalitis in Western countries. Some HSE children carry inborn errors of the Toll-like receptor 3 (TLR3)-dependent IFN-α/β– and -λ–inducing pathway. Induced pluripotent stem cell (iPSC)-derived cortical neurons with TLR3 pathway mutations are highly susceptible to HSV-1, due to impairment of cell-intrinsic TLR3-IFN immunity. In contrast, the contribution of cell-intrinsic immunity of human trigeminal ganglion (TG) neurons remains unclear. Here, we describe efficient in vitro derivation and purification of TG neurons from human iPSCs via a cranial placode intermediate. The resulting TG neurons are of sensory identity and exhibit robust responses to heat (capsaicin), cold (icilin), and inflammatory pain (ATP). Unlike control cortical neurons, both control and TLR3-deficient TG neurons were highly susceptible to HSV-1. However, pretreatment of control TG neurons with poly(I:C) induced the cells into an anti–HSV-1 state. Moreover, both control and TLR3-deficient TG neurons developed resistance to HSV-1 following pretreatment with IFN-β but not IFN-λ. These data indicate that TG neurons are vulnerable to HSV-1 because they require preemptive stimulation of the TLR3 or IFN-α/β receptors to induce antiviral immunity, whereas cortical neurons possess a TLR3-dependent constitutive resistance that is sufficient to block incoming HSV-1 in the absence of prior antiviral signals. The lack of constitutive resistance in TG neurons in vitro is consistent with their exploitation as a latent virus reservoir in vivo. Our results incriminate deficiencies in the constitutive TLR3-dependent response of cortical neurons in the pathogenesis of HSE.

Cerebral organoids are 3D stem cell-derived models that can be utilized to study the human brain. The current consensus is that cerebral organoids consist of cells derived from the neuroectodermal lineage. This limits their value and applicability, as mesodermal-derived microglia are important players in neural development and disease. Remarkably, here we show that microglia can innately develop within a cerebral organoid model and display their characteristic ramified morphology. The transcriptome and response to inflammatory stimulation of these organoid-grown microglia closely mimic the transcriptome and response of adult microglia acutely isolated from post mortem human brain tissue. In addition, organoid-grown microglia mediate phagocytosis and synaptic material is detected inside them. In all, our study characterizes a microglia-containing organoid model that represents a valuable tool for studying the interplay between microglia, macroglia, and neurons in human brain development and disease., Brain organoid models reported to date lack cells of mesodermal origin, such as microglia. Here, the authors demonstrate that mature microglia-like cells are generated within their cerebral organoid model, providing new avenues for studying human microglia in a three-dimensional brain environment.

Multifocal motor neuropathy (MMN) is a rare inflammatory neuropathy characterized by progressive, asymmetric distal limb weakness and conduction block (CB). Clinically MMN is a pure motor neuropathy, which as such can mimic motor neuron disease. GM1-specific IgM antibodies are present in the serum of approximately half of all MMN patients, and are thought to play a key role in the immune pathophysiology. Intravenous immunoglobulin (IVIg) treatment has been shown to be effective in MMN in five randomized placebo-controlled trials. Despite long-term treatment with intravenous immunoglobulin (IVIg), which is efficient in the majority of patients, slowly progressive axonal degeneration and subsequent muscle weakness cannot be fully prevented. In this review, we will discuss the current understanding of the immune pathogenesis underlying MMN and how this may cause CB, available treatment strategies and future therapeutic targets.

Copyright © 2016, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., To elucidate the genetic architecture of amyotrophic lateral sclerosis (ALS) and find associated loci, we assembled a custom imputation reference panel from whole-genome-sequenced patients with ALS and matched controls (n = 1,861). Through imputation and mixed-model association analysis in 12,577 cases and 23,475 controls, combined with 2,579 cases and 2,767 controls in an independent replication cohort, we fine-mapped a new risk locus on chromosome 21 and identified C21orf2 as a gene associated with ALS risk. In addition, we identified MOBP and SCFD1 as new associated risk loci. We established evidence of ALS being a complex genetic trait with a polygenic architecture. Furthermore, we estimated the SNP-based heritability at 8.5%, with a distinct and important role for low-frequency variants (frequency 1-10%). This study motivates the interrogation of larger samples with full genome coverage to identify rare causal variants that underpin ALS risk.

Free tissue transfer has been the gold standard of extensive skull base reconstruction, but the onlay of free flaps onto skull base defects carries the risk of cerebrospinal fluid (CSF) leakage. The purpose of this study was the evaluation of a novel technique of a combined sub- and onlay concept with a partially intracranially positioned folded free fasciocutaneous flap in terms of flap applicability, versatility and complication rate. Within 5 years, 7 patients with anterior (n=4), middle (n=2) or posterior (n=1) skull base defects were reconstructed with free extended lateral arm (n=3) or anterolateral thigh (n=4) flaps. The flaps were partially intracranially positioned and fixed with osteo-dermal sutures. Both flaps proved to be applicable in terms of sealing efficiency, minimizing intracranial flap volume and folding. No flap loss was observed. Specific complications consisted of one pneumocranium via an accessory frontal sinus and one cerebellar herniation due to lumbar CSF loss. No flap failure or haematoma of the intracranial flap part occurred. This new concept of intracranial positioning of fasciocutaneous flaps in a sandwich technique using osteo-dermal sutures should be considered as a primary treatment for skull base reconstruction rather than merely as a salvage manoeuvre.

We investigated the pathogenicity of immunoglobulin M (IgM) anti-GM1 antibodies in serum from patients with multifocal motor neuropathy (MMN) using human induced pluripotent stem cell (iPSC)-derived motor neurons (MNs).iPSCs were generated from fibroblasts and differentiated into MNs. We studied the binding of IgM to MNs, their complement-activating properties, and effects on structural integrity using fluorescence and electron microscopy. Live cell imaging was used to study effects of antibody binding on MNs in the presence and absence of complement.IgM antibody binding to MNs was detected using sera from MMN patients with and without detectable anti-GM1 IgM antibody titers in enzyme-linked immunosorbent assay, but not with sera from (disease) controls. Competition and depletion experiments showed that antibodies specifically bound to GM1 on iPSC-derived MNs. Binding of these antibodies disrupted calcium homeostasis by both complement-dependent and complement-independent pathways. MNs showed marked axonal damage after complement activation, and reduced antibody pathogenicity following treatment with immunoglobulin preparations.Our data provide evidence for the pathogenicity of anti-GM1 IgM antibodies in MMN patients and link their presence to the clinical characteristics of axonal damage and immunoglobulin responsiveness. This iPSC-derived disease model will facilitate diagnosis, studies on autoantibody pathogenicity, drug development, and screening in immune-mediated neuropathies. Ann Neurol 2016;80:71-88.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the loss of lower and upper motor neurons leading to progressive muscle weakness and respiratory insufficiency. No treatment is currently available to cure ALS. Recent progress has led to the identification of several novel genetic determinants of this disease, including repeat expansions in the ataxin-2 (ATXN2) gene. Ataxin-2 is mislocalized in ALS patients and represents a relatively common susceptibility gene in ALS, making it a promising therapeutic target. In this review, we summarize genetic and pathological data implicating ataxin-2 in ALS, discuss potential disease mechanisms linked to altered ataxin-2 localization or function, and propose potential strategies for therapeutic intervention in ALS based on ataxin-2.

Objective: To investigate whether high innate activity of the classical and lectin pathways of complement is associated with multifocal motor neuropathy (MMN) and whether levels of innate complement activity or the potential of anti-GM1 antibodies to activate the complement system correlate with disease severity. Methods: We performed a case-control study including 79 patients with MMN and 79 matched healthy controls. Muscle weakness was documented with Medical Research Council scale sum score and axonal loss with nerve conduction studies. Activity of the classical and lectin pathways of complement was assessed by ELISA. We also determined serum mannose-binding lectin (MBL) concentrations and polymorphisms in the MBL gene (MBL2) and quantified complement-activating properties of anti-GM1 IgM antibodies by ELISA. Results: Activity of the classical and lectin pathways, MBL2 genotypes, and serum MBL concentrations did not differ between patients and controls. Complement activation by anti-GM1 IgM antibodies was exclusively mediated through the classical pathway and correlated with antibody titers (p < 0.001). Logistic regression analysis showed that both high innate activity of the classical pathway of complement and high complement-activating capacity of anti-GM1 IgM antibodies were significantly associated with more severe muscle weakness and axonal loss. Conclusion: High innate activity of the classical pathway of complement and efficient complement-activating properties of anti-GM1 IgM antibodies are determinants of disease severity in patients with MMN. These findings underline the importance of anti-GM1 antibody–mediated complement activation in the pathogenesis and clinical course of MMN.