Microglial over-pruning of synapses during development in autism-associated SCN2A-deficient mice and human cerebral organoids.

Autism spectrum disorder (ASD) is a major neurodevelopmental disorder affecting 1 in 36 children in the United States. While neurons have been the focus of understanding ASD, an altered neuro-immune response in the brain may be closely associated with ASD, and a neuro-immune interaction could play a role in the disease progression. As the resident immune cells of the brain, microglia regulate brain development and homeostasis via core functions including phagocytosis of synapses. While ASD has been traditionally considered a polygenic disorder, recent large-scale human genetic studies have identified SCN2A deficiency as a leading monogenic cause of ASD and intellectual disability. We generated a Scn2a-deficient mouse model, which displays major behavioral and neuronal phenotypes. However, the role of microglia in this disease model is unknown. Here, we reported that Scn2a-deficient mice have impaired learning and memory, accompanied by reduced synaptic transmission and lower spine density in neurons of the hippocampus. Microglia in Scn2a-deficient mice are partially activated, exerting excessive phagocytic pruning of post-synapses related to the complement C3 cascades during selective developmental stages. The ablation of microglia using PLX3397 partially restores synaptic transmission and spine density. To extend our findings from rodents to human cells, we established a microglia-incorporated human cerebral organoid model carrying an SCN2A protein-truncating mutation identified in children with ASD. We found that human microglia display increased elimination of post-synapse in cerebral organoids carrying the SCN2A mutation. Our study establishes a key role of microglia in multi-species autism-associated models of SCN2A deficiency from mouse to human cells.

Emerging Human Pluripotent Stem Cell-Based Human-Animal Brain Chimeras for Advancing Disease Modeling and Cell Therapy for Neurological Disorders.

Human pluripotent stem cell (hPSC) models provide unprecedented opportunities to study human neurological disorders by recapitulating human-specific disease mechanisms. In particular, hPSC-based human-animal brain chimeras enable the study of human cell pathophysiology in vivo. In chimeric brains, human neural and immune cells can maintain human-specific features, undergo maturation, and functionally integrate into host brains, allowing scientists to study how human cells impact neural circuits and animal behaviors. The emerging human-animal brain chimeras hold promise for modeling human brain cells and their interactions in health and disease, elucidating the disease mechanism from molecular and cellular to circuit and behavioral levels, and testing the efficacy of cell therapy interventions. Here, we discuss recent advances in the generation and applications of using human-animal chimeric brain models for the study of neurological disorders, including disease modeling and cell therapy.

Human iPSC-derived microglia sense and dampen hyperexcitability of cortical neurons carrying the epilepsy-associated SCN2A-L1342P mutation.

Neuronal hyperexcitability is a hallmark of seizures. It has been recently shown in rodent models of seizures that microglia, the brain's resident immune cells, can respond to and modulate neuronal excitability. However, how human microglia interacts with human neurons to regulate hyperexcitability mediated by epilepsy-causing genetic mutation found in human patients remains unknown. The SCN2A genetic locus is responsible for encoding the voltage-gated sodium channel Nav1.2, recognized as one of the leading contributors to monogenic epilepsies. Previously, we demonstrated that the recurring Nav1.2-L1342P mutation identified in patients with epilepsy leads to hyperexcitability in a hiPSC-derived cortical neuron model from a male donor. While microglia play an important role in the brain, these cells originate from a different lineage (yolk sac) and thus are not naturally present in hiPSCs-derived neuronal culture. To study how microglia respond to diseased neurons and influence neuronal excitability, we established a co-culture model comprising hiPSC-derived neurons and microglia. We found that microglia display altered morphology with increased branch length and enhanced calcium signal when co-cultured with neurons carrying the Nav1.2-L1342P mutation. Moreover, the presence of microglia significantly lowers the action potential firing of neurons carrying the mutation. Interestingly, we further demonstrated that the current density of sodium channels in neurons carrying the epilepsy-associated mutation was reduced in the presence of microglia. Taken together, our work reveals a critical role of human iPSCs-derived microglia in sensing and dampening hyperexcitability mediated by an epilepsy-causing mutation present in human neurons, highlighting the importance of neuron-microglia interactions in human pathophysiology.

Microglial over-pruning of synapses during development in autism-associated SCN2A-deficient mice and human cerebral organoids.

Autism spectrum disorder (ASD) is a major neurodevelopmental disorder affecting 1 in 36 children in the United States. While neurons have been the focus to understand ASD, an altered neuro-immune response in the brain may be closely associated with ASD, and a neuro-immune interaction could play a role in the disease progression. As the resident immune cells of the brain, microglia regulate brain development and homeostasis via core functions including phagocytosis of synapses. While ASD has been traditionally considered a polygenic disorder, recent large-scale human genetic studies have identified SCN2A deficiency as a leading monogenic cause of ASD and intellectual disability. We generated a Scn2a-deficient mouse model, which displays major behavioral and neuronal phenotypes. However, the role of microglia in this disease model is unknown. Here, we reported that Scn2a-deficient mice have impaired learning and memory, accompanied by reduced synaptic transmission and lower spine density in neurons of the hippocampus. Microglia in Scn2a-deficient mice are partially activated, exerting excessive phagocytic pruning of post-synapses related to the complement C3 cascades during selective developmental stages. The ablation of microglia using PLX3397 partially restores synaptic transmission and spine density. To extend our findings from rodents to human cells, we established a microglial-incorporated human cerebral organoid model carrying an SCN2A protein-truncating mutation identified in children with ASD. We found that human microglia display increased elimination of post-synapse in cerebral organoids carrying the SCN2A mutation. Our study establishes a key role of microglia in multi-species autism-associated models of SCN2A deficiency from mouse to human cells.

Analyses of the autism-associated neuroligin-3 R451C mutation in human neurons reveal a gain-of-function synaptic mechanism.

Mutations in many synaptic genes are associated with autism spectrum disorders (ASD), suggesting that synaptic dysfunction is a key driver of ASD pathogenesis. Among these mutations, the R451C substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs. In mice, the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities, but the impact of the NLGN3 R451C mutation on human neurons has not been investigated. Here, we generated human knockin neurons with the NLGN3 R451C and NLGN3 null mutations. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses; meanwhile NLGN3 knockout neurons showed reduction in excitatory synaptic strengths. Moreover, overexpression of NLGN3 R451C recapitulated the synaptic enhancement in human neurons. Notably, the augmentation of excitatory transmission was confirmed in vivo with human neurons transplanted into mouse forebrain. Using single-cell RNA-seq experiments with co-cultured excitatory and inhibitory NLGN3 R451C-mutant neurons, we identified differentially expressed genes in relatively mature human neurons corresponding to synaptic gene expression networks. Moreover, gene ontology and enrichment analyses revealed convergent gene networks associated with ASDs and other mental disorders. Our findings suggest that the NLGN3 R451C mutation induces a gain-of-function enhancement in excitatory synaptic transmission that may contribute to the pathophysiology of ASD.

Type-I-interferon signaling drives microglial dysfunction and senescence in human iPSC models of Down syndrome and Alzheimer's disease.

Microglia are critical in brain development and Alzheimer's disease (AD) etiology. Down syndrome (DS) is the most common genetic developmental disorder and risk factor for AD. Surprisingly, little information is available on the impact of trisomy of human chromosome 21 (Hsa21) on microglial functions during DS brain development and in AD in DS. Using induced pluripotent stem cell (iPSC)-based organoid and chimeric mouse models, we report that DS microglia exhibit an enhanced synaptic pruning function, which alters neuronal synaptic functions. In response to human brain tissue-derived pathological tau, DS microglia undergo cellular senescence and exhibit elevated type-I-interferon signaling. Mechanistically, knockdown of Hsa21-encoded type I interferon receptors, IFNARs, rescues the DS microglial phenotypes both during brain development and in response to pathological tau. Our findings provide in vivo evidence that human microglia respond to pathological tau by exhibiting dystrophic phenotypes. Targeting IFNARs may improve DS microglial functions and prevent senescence.

Developing human pluripotent stem cell-based cerebral organoids with a controllable microglia ratio for modeling brain development and pathology.

Microglia play critical roles in brain development, homeostasis, and disease. Microglia in animal models cannot accurately model human microglia due to notable transcriptomic and functional differences between human and other animal microglia. Incorporating human pluripotent stem cell (hPSC)-derived microglia into brain organoids provides unprecedented opportunities to study human microglia. However, an optimized method that integrates appropriate amounts of microglia into brain organoids at a proper time point, resembling in vivo brain development, is still lacking. Here, we report a new brain region-specific, microglia-containing organoid model by co-culturing hPSC-derived primitive neural progenitor cells and primitive macrophage progenitors. In the organoids, the number of human microglia can be controlled, and microglia exhibit phagocytic activity and synaptic pruning function. Furthermore, human microglia respond to Zika virus infection of the organoids. Our findings establish a new microglia-containing brain organoid model that will serve to study human microglial function in a variety of neurological disorders.

High-Fidelity Modeling of Human Microglia with Pluripotent Stem Cells.

Gosselin et al. (2017) reported that a tissue-environment-dependent transcriptional network specifies human microglia identity and that in vitro environments drastically alter the human microglia transcriptome. Recent 3-dimensional culture and human-mouse chimeric brain modeling systems developed using human pluripotent stem cells help us understand the complex properties of human microglia.

Human iPSC-derived mature microglia retain their identity and functionally integrate in the chimeric mouse brain.

Microglia, the brain-resident macrophages, exhibit highly dynamic functions in neurodevelopment and neurodegeneration. Human microglia possess unique features as compared to mouse microglia, but our understanding of human microglial functions is largely limited by an inability to obtain human microglia under homeostatic states. Here, we develop a human pluripotent stem cell (hPSC)-based microglial chimeric mouse brain model by transplanting hPSC-derived primitive macrophage progenitors into neonatal mouse brains. Single-cell RNA-sequencing of the microglial chimeric mouse brains reveals that xenografted hPSC-derived microglia largely retain human microglial identity, as they exhibit signature gene expression patterns consistent with physiological human microglia and recapitulate heterogeneity of adult human microglia. Importantly, the engrafted hPSC-derived microglia exhibit dynamic response to cuprizone-induced demyelination and species-specific transcriptomic differences in the expression of neurological disease-risk genes in microglia. This model will serve as a tool to study the role of human microglia in brain development and degeneration.

Pluripotent Stem Cell-Derived Cerebral Organoids Reveal Human Oligodendrogenesis with Dorsal and Ventral Origins.

The process of oligodendrogenesis has been relatively well delineated in the rodent brain. However, it remains unknown whether analogous developmental processes are manifested in the human brain. Here we report oligodendrogenesis in forebrain organoids, generated by using OLIG2-GFP knockin human pluripotent stem cell (hPSC) reporter lines. OLIG2/GFP exhibits distinct temporal expression patterns in ventral forebrain organoids (VFOs) versus dorsal forebrain organoids (DFOs). Interestingly, oligodendrogenesis can be induced in both VFOs and DFOs after neuronal maturation. Assembling VFOs and DFOs to generate fused forebrain organoids (FFOs) promotes oligodendroglia maturation. Furthermore, dorsally derived oligodendroglial cells outcompete ventrally derived oligodendroglia and become dominant in FFOs after long-term culture. Thus, our organoid models reveal human oligodendrogenesis with ventral and dorsal origins. These models will serve to study the phenotypic and functional differences between human ventrally and dorsally derived oligodendroglia and to reveal mechanisms of diseases associated with cortical myelin defects.

OLIG2 Drives Abnormal Neurodevelopmental Phenotypes in Human iPSC-Based Organoid and Chimeric Mouse Models of Down Syndrome.

Down syndrome (DS) is a common neurodevelopmental disorder, and cognitive defects in DS patients may arise from imbalances in excitatory and inhibitory neurotransmission. Understanding the mechanisms underlying such imbalances may provide opportunities for therapeutic intervention. Here, we show that human induced pluripotent stem cells (hiPSCs) derived from DS patients overproduce OLIG2+ ventral forebrain neural progenitors. As a result, DS hiPSC-derived cerebral organoids excessively produce specific subclasses of GABAergic interneurons and cause impaired recognition memory in neuronal chimeric mice. Increased OLIG2 expression in DS cells directly upregulates interneuron lineage-determining transcription factors. shRNA-mediated knockdown of OLIG2 largely reverses abnormal gene expression in early-stage DS neural progenitors, reduces interneuron production in DS organoids and chimeric mouse brains, and improves behavioral deficits in DS chimeric mice. Thus, altered OLIG2 expression may underlie neurodevelopmental abnormalities and cognitive defects in DS patients.

Three-Dimensional Hyaluronic Acid Hydrogel-Based Models for In Vitro Human iPSC-Derived NPC Culture and Differentiation.

Human induced pluripotent stem cell-derived neural progenitor cells (hiPSC-NPCs) are considered as a promising cell source for transplantation and have been used for organoid fabrication to recapitulate central nervous system (CNS) diseases in vitro. The establishment of three-dimensional (3D) in vitro model with hiPSC-NPCs and control of their differentiation is significantly critical for understanding biological processes and CNS disease and regeneration. Here we implemented 3D methacrylated hyaluronic acid (Me-HA) hydrogels with encapsulation of hiPSC-NPCs as in vitro culture models and further investigated the role of the hydrogel rigidity on the cell behavior of hiPSC-NPCs. We first encapsulated single dispersive hiPSC-NPCs within both soft and stiff Me-HA hydrogel and found that hiPSC-NPCs gradually self-assembled and aggregated to form 3D spheroids. Then, the hiPSC-NPCs were laden into Me-HA hydrogels in the form of spheroids to evaluate their spontaneous differentiation in response to hydrogel rigidity. The soft Me-HA hydrogel-encapsulated hiPSC-NPCs displayed robust neurite outgrowth and showed high levels of spontaneous neural differentiation. We further encapsulated Down Syndrome (DS) patient-specific hiPSC-derived NPCs (DS-NPCs) spheroids within our hydrogels. DS-NPCs remained excellent cell viability in both soft and stiff Me-HA hydrogels. Similarly, soft hydrogels promoted neural differentiation of DS-NPCs by significantly upregulating neural maturation markers. This study demonstrates that soft matrix promotes neural differentiation of hiPSC-NPCs and HA-based hydrogels with hiPSC-NPCs or DS-NPCs are effective 3D models for CNS disease study.

Generating CNS organoids from human induced pluripotent stem cells for modeling neurological disorders.

Understanding human brain development and disease is largely hampered by the relative inaccessibility of human brain tissues. Recent advances in human induced pluripotent stem cells (hiPSCs) have led to the generation of unlimited human neural cells and thereby facilitate the investigation of human brain development and pathology. Compared with traditional 2-dimensional (2D) culture methods, culturing the hiPSC-derived neural cells in a three-dimensional (3D) free-floating manner generates human central nervous system (CNS) organoids. These 3D CNS organoids possess the unique advantage of recapitulating multi-regional or region-specific cytoarchitecture seen in the early human fetal brain development. The CNS organoids are becoming a strong complement to the animal model in studying brain development and pathology, and developing new therapies to treat neurodevelopmental diseases. Further improvements to the long-term maintenance and neural maturation of the organoids may allow them to model neurodegenerative diseases. In this review, we will summarize the current development of hiPSCs to generate CNS organoids for modeling neurological disorders and future perspective.

Protease Omi facilitates neurite outgrowth in mouse neuroblastoma N2a cells by cleaving transcription factor E2F1.

AIM: Omi is an ATP-independent serine protease that is necessary for neuronal function and survival. The aim of this study was to investigate the role of protease Omi in regulating differentiation of mouse neuroblastoma cells and to identify the substrate of Omi involved in this process. METHODS: Mouse neuroblastoma N2a cells and Omi protease-deficient mnd2 mice were used in this study. To modulate Omi and E2F1 expression, N2a cells were transfected with expression plasmids, shRNA plasmids or siRNA. Protein levels were detected using immunoblot assays. The interaction between Omi and E2F1 was studied using immunoprecipitation, GST pulldown and in vitro cleavage assays. N2a cells were treated with 20 µmol/L retinoic acid (RA) and 1% fetal bovine serum to induce neurite outgrowth, which was measured using Image J software. RESULTS: E2F1 was significantly increased in Omi knockdown cells and in brain lysates of mnd2 mice, and was decreased in cells overexpressing wild-type Omi, but not inactive Omi S276C. In brain lysates of mnd2 mice, endogenous E2F1 was co-immunoprecipitated with endogenous Omi. In vitro cleavage assay demonstrated that Omi directly cleaved E2F1. Treatment of N2a cells with RA induced marked differentiation and neurite outgrowth accompanied by significantly increased Omi and decreased E2F1 levels, which were suppressed by pretreatment with the specific Omi inhibitor UCF-101. Knockdown of Omi in N2a cells suppressed RA-induced neurite outgrowth, which was partially restored by knockdown of E2F1. CONCLUSION: Protease Omi facilitates neurite outgrowth by cleaving the transcription factor E2F1 in differentiated neuroblastoma cells; E2F1 is a substrate of Omi.

Bcl-2-dependent upregulation of autophagy by sequestosome 1/p62 in vitro.

AIM: To investigate whether sequestosome 1/p62 (p62), a key cargo adaptor protein involved in both the ubiquitin-proteasome system and the autophagy-lysosome system, could directly regulate autophagy in vitro. METHODS: HEK 293 cells or HeLa cells were transfected with p62-expressing plasmids or siRNA targeting p62. The cells or the cell lysates were subsequently subjected to immunofluorescence assay, immunoprecipitation assay, or immunoblot analysis. In vitro pulldown assay was used to study the interaction of p62 with Bcl-2. RESULTS: Overexpression of p62 significantly increased the basal level of autophagy in both HEK 293 cells and HeLa cells, whereas knockdown of p62 significantly decreased the basal level of autophagy. In vitro pulldown assay showed that p62 directly interacted with Bcl-2. It was observed in HeLa cells that p62 co-localized with Bcl-2. Furthermore, knockdown of p62 in HEK 293 cells significantly increased the amount of Beclin 1 that co-immunoprecipitated with Bcl-2. CONCLUSION: p62 induces autophagy by disrupting the association between Bcl-2 and Beclin 1.

The protease Omi cleaves the mitogen-activated protein kinase kinase MEK1 to inhibit microglial activation.

Inflammation in Parkinson's disease is closely associated with disease pathogenesis. Mutations in Omi, which encodes the protease Omi, are linked to neurodegeneration and Parkinson's disease in humans and in mouse models. The severe neurodegeneration and neuroinflammation that occur in mnd2 (motor neuron degeneration 2) mice result from loss of the protease activity of Omi by the point mutation S276C; however, the substrates of Omi that induce neurodegeneration are unknown. We showed that Omi was required for the production of inflammatory molecules by microglia, which are the resident macrophages in the central nervous system. Omi suppressed the activation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1 and 2 (ERK1/2) by cleaving the upstream kinase MEK1 (mitogen-activated or extracellular signal-regulated protein kinase kinase 1). Knockdown of Omi in microglial cell lines led to activation of ERK1/2 and resulted in degradation of IκBα [α inhibitor of nuclear factor κB (NF-κB)], resulting in NF-κB activation and the expression of genes encoding inflammatory molecules, such as tumor necrosis factor-α and inducible nitric oxide synthase. The production of inflammatory molecules induced by the knockdown of Omi was blocked by the MEK1-specific inhibitor U0126. Furthermore, expression of the protease-deficient S276C Omi mutant in a microglial cell line had no effect on MEK1 cleavage or ERK1/2 activation. In the brains of mnd2 mice, we observed increased transcription of several genes encoding inflammatory molecules, as well as activation of astrocytes and microglia. Therefore, our study demonstrates that Omi is an intrinsic cellular factor that inhibits neuroinflammation.

Hax-1 is rapidly degraded by the proteasome dependent on its PEST sequence.

BACKGROUND: HS-1-associated protein X-1 (Hax-1), is a multifunctional protein that has sequence homology to Bcl-2 family members. HAX-1 knockout animals reveal that it plays an essential protective role in the central nervous system against various stresses. Homozygous mutations in the HAX-1 gene are associated with autosomal recessive forms of severe congenital neutropenia along with neurological symptoms. The protein level of Hax-1 has been shown to be regulated by cellular protease cleavage or by transcriptional suppression upon stimulation. RESULTS: Here, we report a novel post-translational mechanism for regulation of Hax-1 levels in mammalian cells. We identified that PEST sequence, a sequence rich in proline, glutamic acid, serine and threonine, is responsible for its poly-ubiquitination and rapid degradation. Hax-1 is conjugated by K48-linked ubiquitin chains and undergoes a fast turnover by the proteasome system. A deletion mutant of Hax-1 that lacks the PEST sequence is more resistant to the proteasomal degradation and exerts more protective effects against apoptotic stimuli than wild type Hax-1. CONCLUSION: Our data indicate that Hax-1 is a short-lived protein and that its PEST sequence dependent fast degradation by the proteasome may contribute to the rapid cellular responses upon different stimulations.