Myeloid cell replacement is neuroprotective in chronic experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease characterized by demyelination of the central nervous system (CNS). Autologous hematopoietic cell transplantation (HCT) shows promising benefits for relapsing-remitting MS in open-label clinical studies, but the cellular mechanisms underlying its therapeutic effects remain unclear. Using single-nucleus RNA sequencing, we identify a reactive myeloid cell state in chronic experimental autoimmune encephalitis (EAE) associated with neuroprotection and immune suppression. HCT in EAE mice results in an increase of the neuroprotective myeloid state, improvement of neurological deficits, reduced number of demyelinated lesions, decreased number of effector T cells and amelioration of reactive astrogliosis. Enhancing myeloid cell incorporation after a modified HCT further improved these neuroprotective effects. These data suggest that myeloid cell manipulation or replacement may be an effective therapeutic strategy for chronic inflammatory conditions of the CNS.

Generation of human excitatory forebrain neurons by cooperative binding of proneural NGN2 and homeobox factor EMX1.

Generation of defined neuronal subtypes from human pluripotent stem cells remains a challenge. The proneural factor NGN2 has been shown to overcome experimental variability observed by morphogen-guided differentiation and directly converts pluripotent stem cells into neurons, but their cellular heterogeneity has not been investigated yet. Here, we found that NGN2 reproducibly produces three different kinds of excitatory neurons characterized by partial coactivation of other neurotransmitter programs. We explored two principle approaches to achieve more precise specification: prepatterning the chromatin landscape that NGN2 is exposed to and combining NGN2 with region-specific transcription factors. Unexpectedly, the chromatin context of regionalized neural progenitors only mildly altered genomic NGN2 binding and its transcriptional response and did not affect neurotransmitter specification. In contrast, coexpression of region-specific homeobox factors such as EMX1 resulted in drastic redistribution of NGN2 including recruitment to homeobox targets and resulted in glutamatergic neurons with silenced nonglutamatergic programs. These results provide the molecular basis for a blueprint for improved strategies for generating a plethora of defined neuronal subpopulations from pluripotent stem cells for therapeutic or disease-modeling purposes.

Biphasic regulation of epigenetic state by matrix stiffness during cell reprogramming.

We investigate how matrix stiffness regulates chromatin reorganization and cell reprogramming and find that matrix stiffness acts as a biphasic regulator of epigenetic state and fibroblast-to-neuron conversion efficiency, maximized at an intermediate stiffness of 20 kPa. ATAC sequencing analysis shows the same trend of chromatin accessibility to neuronal genes at these stiffness levels. Concurrently, we observe peak levels of histone acetylation and histone acetyltransferase (HAT) activity in the nucleus on 20 kPa matrices, and inhibiting HAT activity abolishes matrix stiffness effects. G-actin and cofilin, the cotransporters shuttling HAT into the nucleus, rises with decreasing matrix stiffness; however, reduced importin-9 on soft matrices limits nuclear transport. These two factors result in a biphasic regulation of HAT transport into nucleus, which is directly demonstrated on matrices with dynamically tunable stiffness. Our findings unravel a mechanism of the mechano-epigenetic regulation that is valuable for cell engineering in disease modeling and regenerative medicine applications.

Stress response silencing by an E3 ligase mutated in neurodegeneration.

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health1-3. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.

RAPIDASH: A tag-free enrichment of ribosome-associated proteins reveals compositional dynamics in embryonic tissues and stimulated macrophages.

Ribosomes are emerging as direct regulators of gene expression, with ribosome-associated proteins (RAPs) allowing ribosomes to modulate translational control. However, a lack of technologies to enrich RAPs across many sample types has prevented systematic analysis of RAP number, dynamics, and functions. Here, we have developed a label-free methodology called RAPIDASH to enrich ribosomes and RAPs from any sample. We applied RAPIDASH to mouse embryonic tissues and identified hundreds of potential RAPs, including DHX30 and LLPH, two forebrain RAPs important for neurodevelopment. We identified a critical role of LLPH in neural development that is linked to the translation of genes with long coding sequences. Finally, we characterized ribosome composition remodeling during immune activation and observed extensive changes post-stimulation. RAPIDASH has therefore enabled the discovery of RAPs ranging from those with neuroregulatory functions to those activated by immune stimuli, thereby providing critical insights into how ribosomes are remodeled.

Integrative analyses highlight functional regulatory variants associated with neuropsychiatric diseases.

Noncoding variants of presumed regulatory function contribute to the heritability of neuropsychiatric disease. A total of 2,221 noncoding variants connected to risk for ten neuropsychiatric disorders, including autism spectrum disorder, attention deficit hyperactivity disorder, bipolar disorder, borderline personality disorder, major depression, generalized anxiety disorder, panic disorder, post-traumatic stress disorder, obsessive-compulsive disorder and schizophrenia, were studied in developing human neural cells. Integrating epigenomic and transcriptomic data with massively parallel reporter assays identified differentially-active single-nucleotide variants (daSNVs) in specific neural cell types. Expression-gene mapping, network analyses and chromatin looping nominated candidate disease-relevant target genes modulated by these daSNVs. Follow-up integration of daSNV gene editing with clinical cohort analyses suggested that magnesium transport dysfunction may increase neuropsychiatric disease risk and indicated that common genetic pathomechanisms may mediate specific symptoms that are shared across multiple neuropsychiatric diseases.

Insights and applications of direct neuronal reprogramming.

Direct neuronal reprogramming converts somatic cells of a defined lineage into induced neuronal cells without going through a pluripotent intermediate. This approach not only provides access to the otherwise largely inaccessible cells of the brain for neuronal disease modeling, but also holds great promise for ultimately enabling neuronal cell replacement without the use of transplantation. To improve efficiency and specificity of direct neuronal reprogramming, much of the current efforts aim to understand the mechanisms that safeguard cell identities and how the reprogramming cells overcome the barriers resisting fate changes. Here, we review recent discoveries into the mechanisms by which the donor cell program is silenced, and new cell identities are established. We also discuss advancements that have been made toward fine-tuning the output of these reprogramming systems to generate specific types of neuronal cells. Finally, we highlight the benefit of using direct neuronal reprogramming to study age-related disorders and the potential of in vivo direct reprogramming in regenerative medicine.

Ascl1 and Ngn2 convert mouse embryonic stem cells to neurons via functionally distinct paths.

Ascl1 and Ngn2, closely related proneural transcription factors, are able to convert mouse embryonic stem cells into induced neurons. Despite their similarities, these factors elicit only partially overlapping transcriptional programs, and it remains unknown whether cells are converted via distinct mechanisms. Here we show that Ascl1 and Ngn2 induce mutually exclusive side populations by binding and activating distinct lineage drivers. Furthermore, Ascl1 rapidly dismantles the pluripotency network and installs neuronal and trophoblast cell fates, while Ngn2 generates a neural stem cell-like intermediate supported by incomplete shutdown of the pluripotency network. Using CRISPR-Cas9 knockout screening, we find that Ascl1 relies more on factors regulating pluripotency and the cell cycle, such as Tcf7l1. In the absence of Tcf7l1, Ascl1 still represses core pluripotency genes but fails to exit the cell cycle. However, overexpression of Cdkn1c induces cell cycle exit and restores the generation of neurons. These findings highlight that cell type conversion can occur through two distinct mechanistic paths, even when induced by closely related transcription factors.

Efficient generation of functional neurons from mouse embryonic stem cells via neurogenin-2 expression.

The production of induced neuronal (iN) cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells by the forced expression of proneural transcription factors is rapid, efficient and reproducible. The ability to generate large numbers of human neurons in such a robust manner enables large-scale studies of human neural differentiation and neuropsychiatric diseases. Surprisingly, similar transcription factor-based approaches for converting mouse ESCs into iN cells have been challenging, primarily because of low cell survival. Here, we provide a detailed approach for the efficient and reproducible generation of functional iN cells from mouse ESC cultures by the genetically induced expression of neurogenin-2. The resulting iN cells display mature pre- and postsynaptic specializations and form synaptic networks. Our method provides the basis for studying neuronal development and enables the direct comparison of cellular phenotypes in mouse and human neurons generated in an equivalent way. The procedure requires 14 d and can be carried out by users with expertise in stem cell culture.

A cell therapy approach to restore microglial Trem2 function in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) remains one of the grand challenges facing human society. Much controversy exists around the complex and multifaceted pathogenesis of this prevalent disease. Given strong human genetic evidence, there is little doubt, however, that microglia play an important role in preventing degeneration of neurons. For example, loss of function of the microglial gene Trem2 renders microglia dysfunctional and causes an early-onset neurodegenerative syndrome, and Trem2 variants are among the strongest genetic risk factors for AD. Thus, restoring microglial function represents a rational therapeutic approach. Here, we show that systemic hematopoietic cell transplantation followed by enhancement of microglia replacement restores microglial function in a Trem2 mutant mouse model of AD.

Proteostasis and lysosomal quality control deficits in Alzheimer's disease neurons.

The role of proteostasis and organelle homeostasis dysfunction in human aging and Alzheimer's disease (AD) remains unclear. Analyzing proteome-wide changes in human donor fibroblasts and their corresponding transdifferentiated neurons (tNeurons), we find aging and AD synergistically impair multiple proteostasis pathways, most notably lysosomal quality control (LQC). In particular, we show that ESCRT-mediated lysosomal repair defects are associated with both sporadic and PSEN1 familial AD. Aging- and AD-linked defects are detected in fibroblasts but highly exacerbated in tNeurons, leading to enhanced neuronal vulnerability, unrepaired lysosomal damage, inflammatory factor secretion and cytotoxicity. Surprisingly, tNeurons from aged and AD donors spontaneously develop amyloid-ß inclusions co-localizing with LQC markers, LAMP1/2-positive lysosomes and proteostasis factors; we observe similar inclusions in brain tissue from AD patients and APP-transgenic mice. Importantly, compounds enhancing lysosomal function broadly ameliorate these AD-associated pathologies. Our findings establish cell-autonomous LQC dysfunction in neurons as a central vulnerability in aging and AD pathogenesis.

A scalable, GMP-compatible, autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa.

Background: Gene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies. Methods: We developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a combined high-efficiency reprogramming and CRISPR-based genetic correction single step to generate genome scar-free, COL7A1 corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off targets, and cancer-driver variants were evaluated by targeted and whole genome sequencing. Findings: iPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT-treated mice. Interpretation: DEBCT successfully overcomes previous roadblocks and represents a robust, scalable, and safe cGMP manufacturing platform for production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds.

Augmentation of a neuroprotective myeloid state by hematopoietic cell transplantation.

Multiple sclerosis (MS) is an autoimmune disease associated with inflammatory demyelination in the central nervous system (CNS). Autologous hematopoietic cell transplantation (HCT) is under investigation as a promising therapy for treatment-refractory MS. Here we identify a reactive myeloid state in chronic experimental autoimmune encephalitis (EAE) mice and MS patients that is surprisingly associated with neuroprotection and immune suppression. HCT in EAE mice leads to an enhancement of this myeloid state, as well as clinical improvement, reduction of demyelinated lesions, suppression of cytotoxic T cells, and amelioration of reactive astrogliosis reflected in reduced expression of EAE-associated gene signatures in oligodendrocytes and astrocytes. Further enhancement of myeloid cell incorporation into the CNS following a modified HCT protocol results in an even more consistent therapeutic effect corroborated by additional amplification of HCT-induced transcriptional changes, underlining myeloid-derived beneficial effects in the chronic phase of EAE. Replacement or manipulation of CNS myeloid cells thus represents an intriguing therapeutic direction for inflammatory demyelinating disease.

The autism risk factor CHD8 is a chromatin activator in human neurons and functionally dependent on the ERK-MAPK pathway effector ELK1.

The chromodomain helicase DNA-binding protein CHD8 is the most frequently mutated gene in autism spectrum disorder. Despite its prominent disease involvement, little is known about its molecular function in the human brain. CHD8 is a chromatin regulator which binds to the promoters of actively transcribed genes through genomic targeting mechanisms which have yet to be fully defined. By generating a conditional loss-of-function and an endogenously tagged allele in human pluripotent stem cells, we investigated the molecular function and the interaction of CHD8 with chromatin in human neurons. Chromatin accessibility analysis and transcriptional profiling revealed that CHD8 functions as a transcriptional activator at its target genes in human neurons. Furthermore, we found that CHD8 chromatin targeting is cell context-dependent. In human neurons, CHD8 preferentially binds at ETS motif-enriched promoters. This enrichment is particularly prominent on the promoters of genes whose expression significantly changes upon the loss of CHD8. Indeed, among the ETS transcription factors, we identified ELK1 as being most highly correlated with CHD8 expression in primary human fetal and adult cortical neurons and most highly expressed in our stem cell-derived neurons. Remarkably, ELK1 was necessary to recruit CHD8 specifically to ETS motif-containing sites. These findings imply that ELK1 and CHD8 functionally cooperate to regulate gene expression and chromatin states at MAPK/ERK target genes in human neurons. Our results suggest that the MAPK/ERK/ELK1 axis potentially contributes to the pathogenesis caused by CHD8 mutations in human neurodevelopmental disorders.

Tip60-mediated H2A.Z acetylation promotes neuronal fate specification and bivalent gene activation.

Cell lineage specification is accomplished by a concerted action of chromatin remodeling and tissue-specific transcription factors. However, the mechanisms that induce and maintain appropriate lineage-specific gene expression remain elusive. Here, we used an unbiased proteomics approach to characterize chromatin regulators that mediate the induction of neuronal cell fate. We found that Tip60 acetyltransferase is essential to establish neuronal cell identity partly via acetylation of the histone variant H2A.Z. Despite its tight correlation with gene expression and active chromatin, loss of H2A.Z acetylation had little effect on chromatin accessibility or transcription. Instead, loss of Tip60 and acetyl-H2A.Z interfered with H3K4me3 deposition and activation of a unique subset of silent, lineage-restricted genes characterized by a bivalent chromatin configuration at their promoters. Altogether, our results illuminate the mechanisms underlying bivalent chromatin activation and reveal that H2A.Z acetylation regulates neuronal fate specification by establishing epigenetic competence for bivalent gene activation and cell lineage transition.

Directly induced human retinal ganglion cells mimic fetal RGCs and are neuroprotective after transplantation in vivo.

Retinal ganglion cell (RGC) replacement therapy could restore vision in glaucoma and other optic neuropathies. We developed a rapid protocol for directly induced RGC (iRGC) differentiation from human stem cells, leveraging overexpression of NGN2. Neuronal morphology and neurite growth were observed within 1 week of induction; characteristic RGC-specific gene expression confirmed identity. Calcium imaging demonstrated γ-aminobutyric acid (GABA)-induced excitation characteristic of immature RGCs. Single-cell RNA sequencing showed more similarities between iRGCs and early-stage fetal human RGCs than retinal organoid-derived RGCs. Intravitreally transplanted iRGCs survived and migrated into host retinas independent of prior optic nerve trauma, but iRGCs protected host RGCs from neurodegeneration. These data demonstrate rapid iRGC generation in vitro into an immature cell with high similarity to human fetal RGCs and capacity for retinal integration after transplantation and neuroprotective function after optic nerve injury. The simplicity of this system may benefit translational studies on human RGCs.

Synaptogenic effect of APP-Swedish mutation in familial Alzheimer's disease.

Mutations in ß-amyloid (Aß) precursor protein (APP) cause familial Alzheimer's disease (AD) probably by enhancing Aß peptides production from APP. An antibody targeting Aß (aducanumab) was approved as an AD treatment; however, some Aß antibodies have been reported to accelerate, instead of ameliorating, cognitive decline in individuals with AD. Using conditional APP mutations in human neurons for perfect isogenic controls and translational relevance, we found that the APP-Swedish mutation in familial AD increased synapse numbers and synaptic transmission, whereas the APP deletion decreased synapse numbers and synaptic transmission. Inhibition of BACE1, the protease that initiates Aß production from APP, lowered synapse numbers, suppressed synaptic transmission in wild-type neurons, and occluded the phenotype of APP-Swedish-mutant neurons. Modest elevations of Aß, conversely, elevated synapse numbers and synaptic transmission. Thus, the familial AD-linked APP-Swedish mutation under physiologically relevant conditions increased synaptic connectivity in human neurons via a modestly enhanced production of Aß. These data are consistent with the relative inefficacy of BACE1 and anti-Aß treatments in AD and the chronic nature of AD pathogenesis, suggesting that AD pathogenesis is not simply caused by overproduction of toxic Aß but rather by a long-term effect of elevated Aß concentrations.

Endocytosis in the axon initial segment maintains neuronal polarity.

Neurons are highly polarized cells that face the fundamental challenge of compartmentalizing a vast and diverse repertoire of proteins in order to function properly1. The axon initial segment (AIS) is a specialized domain that separates a neuron's morphologically, biochemically and functionally distinct axon and dendrite compartments2,3. How the AIS maintains polarity between these compartments is not fully understood. Here we find that in Caenorhabditis elegans, mouse, rat and human neurons, dendritically and axonally polarized transmembrane proteins are recognized by endocytic machinery in the AIS, robustly endocytosed and targeted to late endosomes for degradation. Forcing receptor interaction with the AIS master organizer, ankyrinG, antagonizes receptor endocytosis in the AIS, causes receptor accumulation in the AIS, and leads to polarity deficits with subsequent morphological and behavioural defects. Therefore, endocytic removal of polarized receptors that diffuse into the AIS serves as a membrane-clearance mechanism that is likely to work in conjunction with the known AIS diffusion-barrier mechanism to maintain neuronal polarity on the plasma membrane. Our results reveal a conserved endocytic clearance mechanism in the AIS to maintain neuronal polarity by reinforcing axonal and dendritic compartment membrane boundaries.

Transition to a mesenchymal state in neuroblastoma confers resistance to anti-GD2 antibody via reduced expression of ST8SIA1.

Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.