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Vitamin B12 is critical for hematopoiesis and myelination. Deficiency can cause neurologic deficits including loss of coordination and cognitive decline. However, diagnosis relies on measurement of vitamin B12 in the blood, which may not accurately reflect the concentration in the brain. Using programmable phage display, we identified an autoantibody targeting the transcobalamin receptor (CD320) in a patient with progressive tremor, ataxia, and scanning speech. Anti-CD320 impaired cellular uptake of cobalamin (B12) in vitro by depleting its target from the cell surface. Despite a normal serum concentration, B12 was nearly undetectable in her cerebrospinal fluid (CSF). Immunosuppressive treatment and high-dose systemic B12 supplementation were associated with increased B12 in the CSF and clinical improvement. Optofluidic screening enabled isolation of a patient-derived monoclonal antibody that impaired B12 transport across an in vitro model of the blood-brain barrier (BBB). Autoantibodies targeting the same epitope of CD320 were identified in seven other patients with neurologic deficits of unknown etiology, 6% of healthy controls, and 21.4% of a cohort of patients with neuropsychiatric lupus. In 132 paired serum and CSF samples, detection of anti-CD320 in the blood predicted B12 deficiency in the brain. However, these individuals did not display any hematologic signs of B12 deficiency despite systemic CD320 impairment. Using a genome-wide CRISPR screen, we found that the low-density lipoprotein receptor serves as an alternative B12 uptake pathway in hematopoietic cells. These findings dissect the tissue specificity of B12 transport and elucidate an autoimmune neurologic condition that may be amenable to immunomodulatory treatment and nutritional supplementation.
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Autoanticuerpos , Deficiencia de Vitamina B 12 , Vitamina B 12 , Humanos , Deficiencia de Vitamina B 12/inmunología , Vitamina B 12/sangre , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Femenino , Receptores de Superficie Celular/metabolismo , Antígenos CD/metabolismo , Persona de Mediana Edad , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/sangre , Barrera Hematoencefálica/metabolismo , MasculinoRESUMEN
Several genetic risk factors for Alzheimer's disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells1. However, the relationship between lipid metabolism in glia and Alzheimer's disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimer's disease, we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimer's disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia, fibrillar Aß induces ACSL1 expression, triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally, conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimer's disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors, potentially providing therapeutic strategies for Alzheimer's disease.
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Enfermedad de Alzheimer , Apolipoproteína E4 , Gotas Lipídicas , Microglía , Animales , Femenino , Humanos , Masculino , Ratones , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Células Madre Pluripotentes Inducidas/citología , Gotas Lipídicas/metabolismo , Gotas Lipídicas/patología , Microglía/citología , Microglía/metabolismo , Microglía/patología , Triglicéridos , Proteínas tau , Medios de Cultivo Condicionados , Fosforilación , Predisposición Genética a la EnfermedadRESUMEN
Several genetic risk factors for Alzheimer's Disease (AD) implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells. However, the relationship between lipid metabolism in glia and AD pathology remains poorly understood. Through single-nucleus RNA-sequencing of AD brain tissue, we have identified a microglial state defined by the expression of the lipid droplet (LD) associated enzyme ACSL1 with ACSL1-positive microglia most abundant in AD patients with the APOE4/4 genotype. In human iPSC-derived microglia (iMG) fibrillar Aß (fAß) induces ACSL1 expression, triglyceride synthesis, and LD accumulation in an APOE-dependent manner. Additionally, conditioned media from LD-containing microglia leads to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for AD with microglial LD accumulation and neurotoxic microglial-derived factors, potentially providing novel therapeutic strategies for AD.
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Recent understanding of how the systemic environment shapes the brain throughout life has led to numerous intervention strategies to slow brain ageing1-3. Cerebrospinal fluid (CSF) makes up the immediate environment of brain cells, providing them with nourishing compounds4,5. We discovered that infusing young CSF directly into aged brains improves memory function. Unbiased transcriptome analysis of the hippocampus identified oligodendrocytes to be most responsive to this rejuvenated CSF environment. We further showed that young CSF boosts oligodendrocyte progenitor cell (OPC) proliferation and differentiation in the aged hippocampus and in primary OPC cultures. Using SLAMseq to metabolically label nascent mRNA, we identified serum response factor (SRF), a transcription factor that drives actin cytoskeleton rearrangement, as a mediator of OPC proliferation following exposure to young CSF. With age, SRF expression decreases in hippocampal OPCs, and the pathway is induced by acute injection with young CSF. We screened for potential SRF activators in CSF and found that fibroblast growth factor 17 (Fgf17) infusion is sufficient to induce OPC proliferation and long-term memory consolidation in aged mice while Fgf17 blockade impairs cognition in young mice. These findings demonstrate the rejuvenating power of young CSF and identify Fgf17 as a key target to restore oligodendrocyte function in the ageing brain.
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Envejecimiento , Encéfalo , Líquido Cefalorraquídeo , Células Precursoras de Oligodendrocitos , Oligodendroglía , Animales , Diferenciación Celular/genética , Líquido Cefalorraquídeo/fisiología , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica , Ratones , Células Precursoras de Oligodendrocitos/metabolismo , Oligodendroglía/metabolismoRESUMEN
SignificanceNeurodegenerative diseases are poorly understood and difficult to treat. One common hallmark is lysosomal dysfunction leading to the accumulation of aggregates and other undegradable materials, which cause damage to brain resident cells. Lysosomes are acidic organelles responsible for breaking down biomolecules and recycling their constitutive parts. In this work, we find that the antiinflammatory and neuroprotective compound, discovered via a phenotypic screen, imparts its beneficial effects by targeting the lysosome and restoring its function. This is established using a genome-wide CRISPRi target identification screen and then confirmed using a variety of lysosome-targeted studies. The resulting small molecule from this study represents a potential treatment for neurodegenerative diseases as well as a research tool for the study of lysosomes in disease.
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Antiinflamatorios/farmacología , Lisosomas/efectos de los fármacos , Enfermedades Neurodegenerativas/metabolismo , Animales , Antiinflamatorios/química , Biomarcadores , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Desarrollo de Medicamentos , Perfilación de la Expresión Génica , Humanos , Ratones , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteínas Smad/agonistasRESUMEN
Lysosome dysfunction is a shared feature of rare lysosomal storage diseases and common age-related neurodegenerative diseases. Microglia, the brain-resident macrophages, are particularly vulnerable to lysosome dysfunction because of the phagocytic stress of clearing dying neurons, myelin, and debris. CD22 is a negative regulator of microglial homeostasis in the aging mouse brain, and soluble CD22 (sCD22) is increased in the cerebrospinal fluid of patients with Niemann-Pick type C disease (NPC). However, the role of CD22 in the human brain remains unknown. In contrast to previous findings in mice, here, we show that CD22 is expressed by oligodendrocytes in the human brain and binds to sialic aciddependent ligands on microglia. Using unbiased genetic and proteomic screens, we identify insulin-like growth factor 2 receptor (IGF2R) as the binding partner of sCD22 on human myeloid cells. Targeted truncation of IGF2R revealed that sCD22 docks near critical mannose 6-phosphatebinding domains, where it disrupts lysosomal protein trafficking. Interfering with the sCD22-IGF2R interaction using CD22 blocking antibodies ameliorated lysosome dysfunction in human NPC1 mutant induced pluripotent stem cellderived microglia-like cells without harming oligodendrocytes in vitro. These findings reinforce the differences between mouse and human microglia and provide a candidate microglia-directed immunotherapeutic to treat NPC.
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Microglía , Enfermedad de Niemann-Pick Tipo C , Animales , Humanos , Lisosomas/metabolismo , Macrófagos/metabolismo , Ratones , Microglía/metabolismo , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Proteómica , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismo , Lectina 2 Similar a Ig de Unión al Ácido Siálico/uso terapéuticoRESUMEN
Protein aggregation diseases involve intracellular accumulation or extracellular deposition of certain protein species in neuronal or glial cells, leading to neurodegeneration and shortened lifespan. Prime examples include Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD), which are affected by overlapping or specific aggregation-prone proteins. Mounting evidence suggests that dysfunctional glial cells may be major drivers for some diseases, and when they are not causal factors, they could still significantly exacerbate or alleviate disease progression by playing a plethora of detrimental or beneficial roles. Here we review the diverse functions performed by glial cells in a variety of protein aggregation diseases, highlighting the complexity of the issue and the interconnected relationships between these multifaceted effects.
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Enfermedades Neurodegenerativas/patología , Neuroglía/patología , Agregación Patológica de Proteínas/patología , Animales , Humanos , Agregado de ProteínasRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Microglia become progressively activated and seemingly dysfunctional with age, and genetic studies have linked these cells to the pathogenesis of a growing number of neurodegenerative diseases. Here we report a striking buildup of lipid droplets in microglia with aging in mouse and human brains. These cells, which we call 'lipid-droplet-accumulating microglia' (LDAM), are defective in phagocytosis, produce high levels of reactive oxygen species and secrete proinflammatory cytokines. RNA-sequencing analysis of LDAM revealed a transcriptional profile driven by innate inflammation that is distinct from previously reported microglial states. An unbiased CRISPR-Cas9 screen identified genetic modifiers of lipid droplet formation; surprisingly, variants of several of these genes, including progranulin (GRN), are causes of autosomal-dominant forms of human neurodegenerative diseases. We therefore propose that LDAM contribute to age-related and genetic forms of neurodegeneration.
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Envejecimiento/patología , Encéfalo/patología , Lípidos , Microglía/patología , Animales , Humanos , Inflamación/patología , RatonesRESUMEN
Mutations in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis (ALS). Both toxic gain of function and loss of function pathogenic mechanisms have been proposed. Accruing evidence from mouse knockout studies point to a role for C9ORF72 as a regulator of immune function. To provide further insight into its cellular function, we performed a genome-wide synthetic lethal CRISPR screen in human myeloid cells lacking C9ORF72. We discovered a strong synthetic lethal genetic interaction between C9ORF72 and FIS1, which encodes a mitochondrial membrane protein involved in mitochondrial fission and mitophagy. Mass spectrometry experiments revealed that in C9ORF72 knockout cells, FIS1 strongly bound to a class of immune regulators that activate the receptor for advanced glycation end (RAGE) products and trigger inflammatory cascades. These findings present a novel genetic interactor for C9ORF72 and suggest a compensatory role for FIS1 in suppressing inflammatory signaling in the absence of C9ORF72.
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Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Técnicas de Inactivación de Genes/métodos , Pruebas Genéticas , Humanos , RNA-Seq , Mutaciones Letales Sintéticas/genética , Células U937RESUMEN
Hexanucleotide GGGGCC repeat expansion in C9ORF72 is the most prevalent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism is the aberrant accumulation of dipeptide repeat (DPR) proteins produced by the unconventional translation of expanded RNA repeats. Here, we performed genome-wide CRISPR-Cas9 screens for modifiers of DPR protein production in human cells. We found that DDX3X, an RNA helicase, suppresses the repeat-associated non-AUG translation of GGGGCC repeats. DDX3X directly binds to (GGGGCC)n RNAs but not antisense (CCCCGG)n RNAs. Its helicase activity is essential for the translation repression. Reduction of DDX3X increases DPR levels in C9ORF72-ALS/FTD patient cells and enhances (GGGGCC)n-mediated toxicity in Drosophila. Elevating DDX3X expression is sufficient to decrease DPR levels, rescue nucleocytoplasmic transport abnormalities, and improve survival of patient iPSC-differentiated neurons. This work identifies genetic modifiers of DPR protein production and provides potential therapeutic targets for C9ORF72-ALS/FTD.
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Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/biosíntesis , ARN Helicasas DEAD-box/metabolismo , Demencia Frontotemporal/metabolismo , Animales , Sistemas CRISPR-Cas , Drosophila , Humanos , Biosíntesis de Proteínas/fisiología , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Astrocytes are critical for the development and function of the central nervous system. In developing brains, immature astrocytes undergo morphological, molecular, cellular, and functional changes as they mature. Although the mechanisms that regulate the maturation of other major cell types in the central nervous system such as neurons and oligodendrocytes have been extensively studied, little is known about the cellular and molecular mechanisms that control astrocyte maturation. Here, we identified molecular markers of astrocyte maturation and established an in vitro assay for studying the mechanisms of astrocyte maturation. Maturing astrocytes in vitro exhibit similar molecular changes and represent multiple molecular subtypes of astrocytes found in vivo. Using this system, we found that astrocyte-to-astrocyte contact strongly promotes astrocyte maturation. In addition, secreted signals from microglia, oligodendrocyte precursor cells, or endothelial cells affect a small subset of astrocyte genes but do not consistently change astrocyte maturation. To identify molecular mechanisms underlying astrocyte maturation, we treated maturing astrocytes with molecules that affect the function of tumor-associated genes. We found that a positive feedback loop of heparin-binding epidermal growth factor-like growth factor (HBEGF) and epidermal growth factor receptor (EGFR) signaling regulates astrocytes maturation. Furthermore, HBEGF, EGFR, and tumor protein 53 (TP53) affect the expression of genes important for cilium development, the circadian clock, and synapse function. These results revealed cellular and molecular mechanisms underlying astrocytes maturation with implications for the understanding of glioblastoma.
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Astrocitos/fisiología , Comunicación Celular/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Animales , Astrocitos/ultraestructura , Células Cultivadas , Células Endoteliales/fisiología , Receptores ErbB/genética , Retroalimentación Fisiológica , Genes Relacionados con las Neoplasias/genética , Factor de Crecimiento Similar a EGF de Unión a Heparina/genética , Microglía/fisiología , Oligodendroglía/fisiología , Ratas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Microglia maintain homeostasis in the central nervous system through phagocytic clearance of protein aggregates and cellular debris. This function deteriorates during ageing and neurodegenerative disease, concomitant with cognitive decline. However, the mechanisms of impaired microglial homeostatic function and the cognitive effects of restoring this function remain unknown. We combined CRISPR-Cas9 knockout screens with RNA sequencing analysis to discover age-related genetic modifiers of microglial phagocytosis. These screens identified CD22, a canonical B cell receptor, as a negative regulator of phagocytosis that is upregulated on aged microglia. CD22 mediates the anti-phagocytic effect of α2,6-linked sialic acid, and inhibition of CD22 promotes the clearance of myelin debris, amyloid-ß oligomers and α-synuclein fibrils in vivo. Long-term central nervous system delivery of an antibody that blocks CD22 function reprograms microglia towards a homeostatic transcriptional state and improves cognitive function in aged mice. These findings elucidate a mechanism of age-related microglial impairment and a strategy to restore homeostasis in the ageing brain.
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Envejecimiento/fisiología , Encéfalo/citología , Homeostasis/efectos de los fármacos , Microglía/efectos de los fármacos , Ácido N-Acetilneuramínico/farmacología , Fagocitosis/efectos de los fármacos , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Envejecimiento/efectos de los fármacos , Envejecimiento/genética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Cognición/efectos de los fármacos , Cognición/fisiología , Femenino , Homeostasis/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/citología , Ácido N-Acetilneuramínico/química , Fagocitosis/genética , Análisis de Secuencia de ARN , Lectina 2 Similar a Ig de Unión al Ácido Siálico/genética , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismoRESUMEN
K562 is widely used in biomedical research. It is one of three tier-one cell lines of ENCODE and also most commonly used for large-scale CRISPR/Cas9 screens. Although its functional genomic and epigenomic characteristics have been extensively studied, its genome sequence and genomic structural features have never been comprehensively analyzed. Such information is essential for the correct interpretation and understanding of the vast troves of existing functional genomics and epigenomics data for K562. We performed and integrated deep-coverage whole-genome (short-insert), mate-pair, and linked-read sequencing as well as karyotyping and array CGH analysis to identify a wide spectrum of genome characteristics in K562: copy numbers (CN) of aneuploid chromosome segments at high-resolution, SNVs and indels (both corrected for CN in aneuploid regions), loss of heterozygosity, megabase-scale phased haplotypes often spanning entire chromosome arms, structural variants (SVs), including small and large-scale complex SVs and nonreference retrotransposon insertions. Many SVs were phased, assembled, and experimentally validated. We identified multiple allele-specific deletions and duplications within the tumor suppressor gene FHIT Taking aneuploidy into account, we reanalyzed K562 RNA-seq and whole-genome bisulfite sequencing data for allele-specific expression and allele-specific DNA methylation. We also show examples of how deeper insights into regulatory complexity are gained by integrating genomic variant information and structural context with functional genomics and epigenomics data. Furthermore, using K562 haplotype information, we produced an allele-specific CRISPR targeting map. This comprehensive whole-genome analysis serves as a resource for future studies that utilize K562 as well as a framework for the analysis of other cancer genomes.
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Genoma Humano , Humanos , Células K562 , Cariotipo , Polimorfismo Genético , Secuenciación Completa del GenomaRESUMEN
Large copy number variants (CNVs) in the human genome are strongly associated with common neurodevelopmental, neuropsychiatric disorders such as schizophrenia and autism. Here we report on the epigenomic effects of the prominent large deletion CNVs on chromosome 22q11.2 and on chromosome 1q21.1. We use Hi-C analysis of long-range chromosome interactions, including haplotype-specific Hi-C analysis, ChIP-Seq analysis of regulatory histone marks, and RNA-Seq analysis of gene expression patterns. We observe changes on all the levels of analysis, within the deletion boundaries, in the deletion flanking regions, along chromosome 22q, and genome wide. We detect gene expression changes as well as pronounced and multilayered effects on chromatin states, chromosome folding and on the topological domains of the chromatin, that emanate from the large CNV locus. These findings suggest basic principles of how such large genomic deletions can alter nuclear organization and affect genomic molecular activity.
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Encéfalo/crecimiento & desarrollo , Cromatina/metabolismo , Dosificación de Gen/genética , Trastornos Mentales/genética , Línea Celular , Cromatina/genética , Cromosomas Humanos Par 1/genética , Cromosomas Humanos Par 22/genética , Genoma Humano/genética , HumanosRESUMEN
Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer's disease-associated gene TM2D3 can preferentially influence uptake of amyloid-ß aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Magnetismo/métodos , Fagocitosis/genética , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Regulación de la Expresión Génica , Estudios de Asociación Genética/métodos , Genoma Humano , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Ratones , Células RAW 264.7 , Transducción de Señal/genética , Células U937RESUMEN
Here, we describe approaches using droplet digital polymerase chain reaction (ddPCR) to validate and quantify somatic mosaic events contributed by transposable-element insertions, copy-number variants, and single-nucleotide variants. In the ddPCR assay, sample or template DNA is partitioned into tens of thousands of individual droplets such that when DNA input is low, the vast majority of droplets contains no more than one copy of template DNA. PCR takes place in each individual droplet and produces a fluorescent readout to indicate the presence or absence of the target of interest allowing for the accurate "counting" of the number of copies present in the sample. The number of partitions is large enough to assay somatic mosaic events with frequencies down to less than 1%.
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Variaciones en el Número de Copia de ADN/genética , Elementos Transponibles de ADN/genética , ADN/aislamiento & purificación , Mosaicismo , Reacción en Cadena de la Polimerasa/métodos , ADN/genética , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases.