RESUMEN
Amyotrophic lateral sclerosis is a rapidly progressive and fatal disease. Although astrocytes are increasingly recognized contributors to the underlying pathogenesis, the cellular autonomy and uniformity of astrocyte reactive transformation in different genetic forms of amyotrophic lateral sclerosis remain unresolved. Here we systematically examine these issues by using highly enriched and human induced pluripotent stem cell-derived astrocytes from patients with VCP and SOD1 mutations. We show that VCP mutant astrocytes undergo cell-autonomous reactive transformation characterized by increased expression of complement component 3 (C3) in addition to several characteristic gene expression changes. We then demonstrate that isochronic SOD1 mutant astrocytes also undergo a cell-autonomous reactive transformation, but that this is molecularly distinct from VCP mutant astrocytes. This is shown through transcriptome-wide analyses, identifying divergent gene expression profiles and activation of different key transcription factors in SOD1 and VCP mutant human induced pluripotent stem cell-derived astrocytes. Finally, we show functional differences in the basal cytokine secretome between VCP and SOD1 mutant human induced pluripotent stem cell-derived astrocytes. Our data therefore reveal that reactive transformation can occur cell autonomously in human amyotrophic lateral sclerosis astrocytes and with a striking degree of early molecular and functional heterogeneity when comparing different disease-causing mutations. These insights may be important when considering astrocyte reactivity as a putative therapeutic target in familial amyotrophic lateral sclerosis.
Asunto(s)
Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Esclerosis Amiotrófica Lateral/metabolismo , Astrocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación/genética , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/genéticaRESUMEN
Neuroinflammation has been shown to mediate the pathophysiological response following traumatic brain injury (TBI). Accumulating evidence implicates astrocytes as key immune cells within the central nervous system (CNS), displaying both pro- and anti-inflammatory properties. The aim of this study was to investigate how in vitro human astrocyte cultures respond to cytokines across a concentration range that approximates the aftermath of human TBI. To this end, enriched cultures of human induced pluripotent stem cell (iPSC)-derived astrocytes were exposed to interleukin-1ß (IL-1ß) (1-10,000 pg/mL), IL-4 (1-10,000 pg/mL), IL-6 (100-1,000,000 pg/mL), IL-10 (1-10,000 pg/mL) and tumor necrosis factor (TNF)-α (1-10,000 pg/mL). After 1, 24, 48 and 72 h, cultures were fixed and immunolabeled, and the secretome/supernatant was analyzed at 24, 48, and 72 h using a human cytokine/chemokine 39-plex Luminex assay. Data were compared to previous in vitro studies of neuronal cultures and clinical TBI studies. The secretome revealed concentration-, time- and/or both concentration- and time-dependent production of downstream cytokines (29, 21, and 17 cytokines, respectively, p<0.05). IL-1ß exposure generated the most profound downstream response (27 cytokines), IL-6 and TNF had intermediate responses (13 and 11 cytokines, respectively), whereas IL-4 and IL-10 only led to weak responses over time or in escalating concentration (8 and 8 cytokines, respectively). Notably, expression of IL-1ß, IL-6, and TNF cytokine receptor mRNA was higher in astrocyte cultures than in neuronal cultures. Several secreted cytokines had temporal trajectories, which corresponded to those seen in the aftermath of human TBI. In summary, iPSC-derived astrocyte cultures exposed to cytokine concentrations reflecting those in TBI generated an increased downstream cytokine production, particularly IL-1ß. Although more work is needed to better understand how different cells in the CNS respond to the neuroinflammatory milieu after TBI, our data shows that iPSC-derived astrocytes represent a tractable model to study cytokine stimulation in a cell type-specific manner.
Asunto(s)
Astrocitos/metabolismo , Lesiones Traumáticas del Encéfalo/metabolismo , Citocinas/metabolismo , Citocinas/farmacología , Astrocitos/efectos de los fármacos , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismoRESUMEN
Cytokine mediated inflammation likely plays an important role in secondary pathology after traumatic brain injury (TBI). The aim of this study was to elucidate secondary cytokine responses in an in vitro enriched (>80%) human stem cell-derived neuronal model. We exposed neuronal cultures to pre-determined and clinically relevant pathophysiological levels of tumor necrosis factor-α (TNF), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß), shown to be present in the inflammatory aftermath of TBI. Data from this reductionist human model were then compared with our in vivo data. Human embryonic stem cell (hESC)-derived neurons were exposed to recombinant TNF (1-10,000 pg/mL), IL-1ß (1-10,000 pg/mL), and IL-6 (0.1-1000 ng/mL). After 1, 24, and 72 h, culture supernatant was sampled and analyzed using a human cytokine/chemokine 42-plex Milliplex kit on the Luminex platform. The culture secretome revealed both a dose- and/or time-dependent release of cytokines. The IL-6 and TNF exposure each resulted in significantly increased levels of >10 cytokines over time, while IL-1ß increased the level of C-X-C motif chemokine 10 (CXCL10/IP10) alone. Importantly, these patterns are consistent with our in vivo (human) TBI data, thus validating our human stem cell-derived neuronal platform as a clinically useful reductionist model. Our data cumulatively suggest that IL-6 and TNF have direct actions, while the action of IL-1ß on human neurons likely occurs indirectly through inflammatory cells. The hESC-derived neurons provide a valuable platform to model cytokine mediated inflammation and can provide important insights into the mechanisms of neuroinflammation after TBI.
Asunto(s)
Lesiones Traumáticas del Encéfalo/metabolismo , Citocinas/metabolismo , Inflamación/metabolismo , Células-Madre Neurales/metabolismo , Células Cultivadas , Citocinas/farmacología , Humanos , Células-Madre Neurales/efectos de los fármacosRESUMEN
Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate ubiquitously expressed regulators of RNA processing. To understand the molecular impact of ALS-causing mutations on neuronal development and disease, we analysed transcriptomes during in vitro differentiation of motor neurons (MNs) from human control and patient-specific VCP mutant induced-pluripotent stem cells (iPSCs). We identify increased intron retention (IR) as a dominant feature of the splicing programme during early neural differentiation. Importantly, IR occurs prematurely in VCP mutant cultures compared with control counterparts. These aberrant IR events are also seen in independent RNAseq data sets from SOD1- and FUS-mutant MNs. The most significant IR is seen in the SFPQ transcript. The SFPQ protein binds extensively to its retained intron, exhibits lower nuclear abundance in VCP mutant cultures and is lost from nuclei of MNs in mouse models and human sporadic ALS. Collectively, we demonstrate SFPQ IR and nuclear loss as molecular hallmarks of familial and sporadic ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Neuronas Motoras/metabolismo , Factor de Empalme Asociado a PTB/genética , Empalme del ARN , Médula Espinal/metabolismo , Proteína que Contiene Valosina/genética , Anciano , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Diferenciación Celular , Modelos Animales de Enfermedad , Exones , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Intrones , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Neuronas Motoras/patología , Factor de Empalme Asociado a PTB/metabolismo , Cultivo Primario de Células , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Análisis de Secuencia de ARN , Médula Espinal/patología , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Proteína que Contiene Valosina/metabolismoRESUMEN
Astrocyte responses to neuronal injury may be beneficial or detrimental to neuronal recovery, but the mechanisms that determine these different responses are poorly understood. Here we show that ephrin type-B receptor 1 (EphB1) is upregulated in injured motor neurons, which in turn can activate astrocytes through ephrin-B1-mediated stimulation of signal transducer and activator of transcription-3 (STAT3). Transcriptional analysis shows that EphB1 induces a protective and anti-inflammatory signature in astrocytes, partially linked to the STAT3 network. This is distinct from the response evoked by interleukin (IL)-6 that is known to induce both pro inflammatory and anti-inflammatory processes. Finally, we demonstrate that the EphB1-ephrin-B1 pathway is disrupted in human stem cell derived astrocyte and mouse models of amyotrophic lateral sclerosis (ALS). Our work identifies an early neuronal help-me signal that activates a neuroprotective astrocytic response, which fails in ALS, and therefore represents an attractive therapeutic target.
Asunto(s)
Astrocitos/citología , Interleucina-6/metabolismo , Neuronas/metabolismo , Receptor EphB1/metabolismo , Factor de Transcripción STAT3/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Antiinflamatorios/farmacología , Astrocitos/metabolismo , Axones/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Inflamación , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/metabolismo , Neuroprotección , Nervio Ciático/metabolismo , Transducción de Señal , TranscriptomaRESUMEN
Motor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs) into highly enriched (> 85%) functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP)-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS.