ABSTRACT
Significant efforts are ongoing to develop refined differentiation protocols to generate midbrain dopamine (DA) neurons from pluripotent stem cells for application in disease modeling, diagnostics, drug screening and cell-based therapies for Parkinson's disease. An increased understanding of the timing and molecular mechanisms that promote the generation of distinct subtypes of human midbrain DA during development will be essential for guiding future efforts to generate molecularly defined and subtype-specific DA neurons from pluripotent stem cells. Here, we use droplet-based single-cell RNA sequencing to transcriptionally profile the developing human ventral midbrain (VM) when the DA neurons are generated (6-11â weeks post-conception) and their subsequent differentiation into functional mature DA neurons in primary fetal 3D organoid-like cultures. This approach reveals that 3D cultures are superior to monolayer conditions for their ability to generate and maintain mature DA neurons; hence, they have the potential to be used for studying human VM development. These results provide a unique transcriptional profile of the developing human fetal VM and functionally mature human DA neurons that can be used to guide stem cell-based therapies and disease modeling approaches in Parkinson's disease.
Subject(s)
Parkinson Disease , Pluripotent Stem Cells , Humans , Parkinson Disease/genetics , Parkinson Disease/therapy , Dopaminergic Neurons , Mesencephalon , Cell Differentiation/geneticsABSTRACT
Huntington's disease is a neurodegenerative disorder caused by CAG expansions in the huntingtin (HTT) gene. Modelling Huntington's disease is challenging, as rodent and cellular models poorly recapitulate the disease as seen in ageing humans. To address this, we generated induced neurons through direct reprogramming of human skin fibroblasts, which retain age-dependent epigenetic characteristics. Huntington's disease induced neurons (HD-iNs) displayed profound deficits in autophagy, characterized by reduced transport of late autophagic structures from the neurites to the soma. These neurite-specific alterations in autophagy resulted in shorter, thinner and fewer neurites specifically in HD-iNs. CRISPRi-mediated silencing of HTT did not rescue this phenotype but rather resulted in additional autophagy alterations in control induced neurons, highlighting the importance of wild-type HTT in normal neuronal autophagy. In summary, our work identifies a distinct subcellular autophagy impairment in adult patient derived Huntington's disease neurons and provides a new rationale for future development of autophagy activation therapies.
Subject(s)
Huntington Disease , Neurodegenerative Diseases , Adult , Autophagy/physiology , Humans , Huntingtin Protein/genetics , Huntington Disease/genetics , NeuronsABSTRACT
Ca2+-sensor proteins are generally implicated in insulin release through SNARE interactions. Here, secretagogin, whose expression in human pancreatic islets correlates with their insulin content and the incidence of type 2 diabetes, is shown to orchestrate an unexpectedly distinct mechanism. Single-cell RNA-seq reveals retained expression of the TRP family members in ß-cells from diabetic donors. Amongst these, pharmacological probing identifies Ca2+-permeable transient receptor potential vanilloid type 1 channels (TRPV1) as potent inducers of secretagogin expression through recruitment of Sp1 transcription factors. Accordingly, agonist stimulation of TRPV1s fails to rescue insulin release from pancreatic islets of glucose intolerant secretagogin knock-out(-/-) mice. However, instead of merely impinging on the SNARE machinery, reduced insulin availability in secretagogin-/- mice is due to ß-cell loss, which is underpinned by the collapse of protein folding and deregulation of secretagogin-dependent USP9X deubiquitinase activity. Therefore, and considering the desensitization of TRPV1s in diabetic pancreata, a TRPV1-to-secretagogin regulatory axis seems critical to maintain the structural integrity and signal competence of ß-cells.
Subject(s)
Gene Expression Regulation , Insulin-Secreting Cells/physiology , Proteins/metabolism , Secretagogins/metabolism , TRPV Cation Channels/metabolism , Animals , Cell Survival , Gene Expression Profiling , Humans , Mice , Mice, Knockout , Secretagogins/deficiency , Single-Cell AnalysisABSTRACT
C4BP (C4b-binding protein) is a polymer of seven identical α chains and one unique ß chain synthesized in liver and pancreas. We showed previously that C4BP enhances islet amyloid polypeptide (IAPP) fibril formation in vitro Now we report that polymeric C4BP strongly inhibited lysis of human erythrocytes incubated with monomeric IAPP, whereas no lysis was observed after incubation with preformed IAPP fibrils. In contrast, incubation with the monomeric α-chain of C4BP was less effective. These data indicate that polymeric C4BP with multiple binding sites for IAPP neutralizes lytic activity of IAPP. Furthermore, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Treatment of INS-1 cells and primary rat islets with IAPP also diminished their ability to secrete insulin upon stimulation with glucose, which was reversed in the presence of C4BP. Further, C4BP was internalized together with IAPP into INS-1 cells. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison with IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-ß-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-κB had a similar effect. Taken together, C4BP protects ß-cells from IAPP cytotoxicity by modulating IAPP fibril formation extracellularly and also, after uptake by the cells, by enhancing cholesterol synthesis.
Subject(s)
Cholesterol/biosynthesis , Complement C4b-Binding Protein/metabolism , Gene Expression Regulation/physiology , Insulin-Secreting Cells/metabolism , Islet Amyloid Polypeptide/biosynthesis , Animals , Cell Line, Tumor , Cholesterol Oxidase/metabolism , Humans , Male , Phosphatidylinositol 3-Kinases/metabolism , Rats , Rats, WistarABSTRACT
High blood glucose triggers the release of insulin from pancreatic beta cells, but if chronic, causes cellular stress, partly due to impaired Ca2+ homeostasis. Ca2+ influx is controlled by voltage-gated calcium channels (CaV) and high density of CaV in the plasma membrane could lead to Ca2+ overload. Trafficking of the pore-forming CaVα1 subunit to the plasma membrane is regulated by auxiliary subunits, such as the CaVß2a subunit. This study investigates, using Ca2+ imaging and immunohistochemistry, the role of palmitoylation of CaVß2a in maintaining Ca2+ homeostasis and beta cell function. RNA sequencing data showed that gene expression of human CACNB2, in particular CACNB2A (CaVß2a), is highest in islets when compared to other tissues. Since CaVß2a can be regulated through palmitoylation of its two cysteines, CaVß2a and its mutant form were overexpressed in pancreatic beta cells. Palmitoylated CaVß2a tethered to the plasma membrane and colocalized with CaV1.2 while the mutant form remained in the cytosol. Interestingly, CaVß2a overexpression raised basal intracellular Ca2+ and increased beta cell apoptosis. Our study shows that palmitoylation of CaVß2a is necessary for CaVα1 trafficking to the plasma membrane. However, excessive number of palmitoylated CaVß2a leads to Ca2+ overload and beta cell death.
Subject(s)
Apoptosis/physiology , Calcium Channels, L-Type/metabolism , Calcium Signaling/physiology , Calcium/metabolism , Insulin-Secreting Cells/physiology , Lipoylation/physiology , Animals , Binding Sites , Cell Line , Insulin-Secreting Cells/cytology , Ion Channel Gating/physiology , Protein Binding , Protein Subunits , RatsABSTRACT
Genetic variation can modulate gene expression, and thereby phenotypic variation and susceptibility to complex diseases such as type 2 diabetes (T2D). Here we harnessed the potential of DNA and RNA sequencing in human pancreatic islets from 89 deceased donors to identify genes of potential importance in the pathogenesis of T2D. We present a catalog of genetic variants regulating gene expression (eQTL) and exon use (sQTL), including many long noncoding RNAs, which are enriched in known T2D-associated loci. Of 35 eQTL genes, whose expression differed between normoglycemic and hyperglycemic individuals, siRNA of tetraspanin 33 (TSPAN33), 5'-nucleotidase, ecto (NT5E), transmembrane emp24 protein transport domain containing 6 (TMED6), and p21 protein activated kinase 7 (PAK7) in INS1 cells resulted in reduced glucose-stimulated insulin secretion. In addition, we provide a genome-wide catalog of allelic expression imbalance, which is also enriched in known T2D-associated loci. Notably, allelic imbalance in paternally expressed gene 3 (PEG3) was associated with its promoter methylation and T2D status. Finally, RNA editing events were less common in islets than previously suggested in other tissues. Taken together, this study provides new insights into the complexity of gene regulation in human pancreatic islets and better understanding of how genetic variation can influence glucose metabolism.
Subject(s)
Genomics , Glucose , Transcriptome/physiology , 5'-Nucleotidase/biosynthesis , 5'-Nucleotidase/genetics , Cell Line , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Female , GPI-Linked Proteins/biosynthesis , GPI-Linked Proteins/genetics , Glucose/genetics , Glucose/metabolism , Humans , Islets of Langerhans , Male , RNA Editing/physiology , RNA, Long Noncoding/biosynthesis , RNA, Long Noncoding/genetics , Tetraspanins/biosynthesis , Tetraspanins/genetics , Vesicular Transport Proteins/biosynthesis , Vesicular Transport Proteins/genetics , p21-Activated Kinases/biosynthesis , p21-Activated Kinases/geneticsABSTRACT
We have previously identified transcription factor B1 mitochondrial (TFB1M) as a type 2 diabetes (T2D) risk gene, using human and mouse genetics. To further understand the function of TFB1M and how it is associated with T2D, we created a ß-cell-specific knockout of Tfb1m, which gradually developed diabetes. Prior to the onset of diabetes, ß-Tfb1m(-/-) mice exhibited retarded glucose clearance owing to impaired insulin secretion. ß-Tfb1m(-/-) islets released less insulin in response to fuels, contained less insulin and secretory granules and displayed reduced ß-cell mass. Moreover, mitochondria in Tfb1m-deficient ß-cells were more abundant with disrupted architecture. TFB1M is known to control mitochondrial protein translation by adenine dimethylation of 12S ribosomal RNA (rRNA). Here, we found that the levels of TFB1M and mitochondrial-encoded proteins, mitochondrial 12S rRNA methylation, ATP production and oxygen consumption were reduced in ß-Tfb1m(-/-) islets. Furthermore, the levels of reactive oxygen species (ROS) in response to cellular stress were increased whereas induction of defense mechanisms was attenuated. We also show increased apoptosis and necrosis as well as infiltration of macrophages and CD4(+) cells in the islets. Taken together, our findings demonstrate that Tfb1m-deficiency in ß-cells caused mitochondrial dysfunction and subsequently diabetes owing to combined loss of ß-cell function and mass. These observations reflect pathogenetic processes in human islets: using RNA sequencing, we found that the TFB1M risk variant exhibited a negative gene-dosage effect on islet TFB1M mRNA levels, as well as insulin secretion. Our findings highlight the role of mitochondrial dysfunction in impairments of ß-cell function and mass, the hallmarks of T2D.
Subject(s)
Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Insulin/biosynthesis , Mitochondria/genetics , Mitochondria/metabolism , Transcription Factors/genetics , Animals , Cell Survival/genetics , Disease Models, Animal , Female , Gene Expression , Humans , Inflammation/genetics , Inflammation/metabolism , Insulin-Secreting Cells/metabolism , Islets of Langerhans/metabolism , Islets of Langerhans/pathology , Male , Mice , Mice, Knockout , Mitochondria/ultrastructure , Oxidative Stress , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Transcription Factors/deficiencyABSTRACT
AIMS/HYPOTHESIS: Our aim was to investigate the association between birthweight and latent autoimmune diabetes in adults (LADA), a common diabetes form with features of both type 1 and type 2 diabetes. METHODS: We used data from the Epidemiological Study of Risk Factors for LADA and Type 2 Diabetes (ESTRID), a Swedish population-based study. Eligible for the analysis were 134 incident LADA cases (glutamic acid decarboxylase antibody [GADA] positive), 350 incident type 2 diabetes cases (GADA negative) and 603 randomly selected controls. We present ORs and 95% CIs for LADA and type 2 diabetes in relation to birthweight, adjusted for sex, age, BMI and family history of diabetes. RESULTS: Low birthweight increased the risk of LADA as well as the risk of type 2 diabetes; OR per kg reduction was estimated as 1.52 (95% CI 1.12, 2.08) and 1.58 (1.23, 2.04), respectively. The OR for participants weighing <3 kg compared with ≥4 kg at birth was estimated as 2.38 (1.23, 4.60) for LADA and 2.37 (1.37, 4.10) for type 2 diabetes. A combination of low birthweight (<3 kg) and current overweight (BMI ≥ 25) further augmented the risk: LADA, OR 3.26 (1.69, 6.29); and type 2 diabetes, OR 39.93 (19.27, 82.71). Family history of diabetes had little impact on these estimates. CONCLUSIONS/INTERPRETATION: Our results suggest that low birthweight may be a risk factor for LADA of the same strength as for type 2 diabetes. These findings support LADA, despite its autoimmune component, having an aetiology that includes factors related to type 2 diabetes.
Subject(s)
Diabetes Mellitus, Type 1/epidemiology , Diabetes Mellitus, Type 1/etiology , Diabetes Mellitus, Type 2/epidemiology , Diabetes Mellitus, Type 2/etiology , Infant, Low Birth Weight , Adult , Aged , Autoantibodies , Case-Control Studies , Diabetes Mellitus, Type 1/immunology , Female , Glutamate Decarboxylase/immunology , Humans , Incidence , Male , Middle Aged , Risk , SwedenABSTRACT
CD55 is a glycosylphosphatidylinositol-anchored protein, which inhibits complement activation by acting on the complement C3 convertases. CD55 is widely localized in the cholesterol rich regions of the cell plasma membrane termed membrane rafts. CD55 is attached to these specialized regions via a GPI link on the outer leaflet of the plasma membrane. Membrane rafts anchor many important signaling proteins, which control several cellular functions within the cell. For example, we recently demonstrated that the membrane raft protein and complement inhibitor CD59 also controls insulin secretion by an intracellular mechanism. Therefore, we have in this study aimed at addressing the expression and function of CD55 in pancreatic beta cells. To this end, we observe that CD55 is highly expressed in INS1 832/13 beta cells as well as human pancreatic islets. Diabetic human islets show a tendency for increased expression of CD55 when compared to the healthy controls. Importantly, silencing of CD55 in INS1 832/13 cells does not affect their insulin secretory capacity. On the other hand, silencing of CD55 diminished the intensity of membrane rafts as determined by Atto-SM staining. We hence conclude that CD55 expression is affected by glycemic status in human islets and plays a critical role in maintaining the conserved structure of rafts in pancreatic islets, which is similar to that of the related complement inhibitor CD59. However CD55 does not interfere with insulin secretion in beta cells, which is in sharp contrast to the action of the complement inhibitor CD59.
Subject(s)
CD55 Antigens/physiology , Insulin/metabolism , Islets of Langerhans/metabolism , Membrane Microdomains/metabolism , Animals , CD55 Antigens/genetics , Cell Line , Gene Expression Profiling , Humans , Insulin Secretion , RatsABSTRACT
BACKGROUND: Most colon cancers start with dysregulated Wnt/ß-catenin signalling and remain a major therapeutic challenge. Examining whether HAMLET (human α-lactalbumin made lethal to tumour cells) may be used for colon cancer treatment is logical, based on the properties of the complex and its biological context. OBJECTIVE: To investigate if HAMLET can be used for colon cancer treatment and prevention. Apc(Min)(/+) mice, which carry mutations relevant to hereditary and sporadic human colorectal tumours, were used as a model for human disease. METHOD: HAMLET was given perorally in therapeutic and prophylactic regimens. Tumour burden and animal survival of HAMLET-treated and sham-fed mice were compared. Tissue analysis focused on Wnt/ß-catenin signalling, proliferation markers and gene expression, using microarrays, immunoblotting, immunohistochemistry and ELISA. Confocal microscopy, reporter assay, immunoprecipitation, immunoblotting, ion flux assays and holographic imaging were used to determine effects on colon cancer cells. RESULTS: Peroral HAMLET administration reduced tumour progression and mortality in Apc(Min)(/+) mice. HAMLET accumulated specifically in tumour tissue, reduced ß-catenin and related tumour markers. Gene expression analysis detected inhibition of Wnt signalling and a shift to a more differentiated phenotype. In colon cancer cells with APC mutations, HAMLET altered ß-catenin integrity and localisation through an ion channel-dependent pathway, defining a new mechanism for controlling ß-catenin signalling. Remarkably, supplying HAMLET to the drinking water from the time of weaning also significantly prevented tumour development. CONCLUSIONS: These data identify HAMLET as a new, peroral agent for colon cancer prevention and treatment, especially needed in people carrying APC mutations, where colon cancer remains a leading cause of death.
Subject(s)
Antineoplastic Agents/therapeutic use , Colonic Neoplasms/drug therapy , Lactalbumin/therapeutic use , Oleic Acids/therapeutic use , Administration, Oral , Animals , Biomarkers, Tumor/metabolism , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/prevention & control , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genes, APC , Genetic Markers , Genetic Predisposition to Disease , Kaplan-Meier Estimate , Male , Mice , Mice, Transgenic , Mutation , Survival Rate , Treatment Outcome , Tumor BurdenABSTRACT
Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance (13)C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 µm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 µm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.
Subject(s)
Antineoplastic Agents/pharmacology , Lactalbumin/pharmacology , Oleic Acid/pharmacology , Oleic Acids/pharmacology , Signal Transduction/drug effects , Antineoplastic Agents/chemistry , Cell Death/drug effects , Cell Shape/drug effects , Cell Survival/drug effects , Citric Acid Cycle/drug effects , Gene Expression Profiling , Gene Regulatory Networks , Humans , Jurkat Cells , Lactalbumin/chemistry , Metabolome/drug effects , Oleic Acid/chemistry , Oleic Acids/chemistry , Oligonucleotide Array Sequence Analysis , Transcriptome/drug effectsABSTRACT
Cell therapy for Parkinson's disease has experienced substantial growth in the past decades with several ongoing clinical trials. Despite increasing refinement of differentiation protocols and standardization of the transplanted neural precursors, the transcriptomic analysis of cells in the transplant after its full maturation in vivo has not been thoroughly investigated. Here, we present spatial transcriptomics analysis of fully differentiated grafts in their host tissue. Unlike earlier transcriptomics analyses using single-cell technologies, we observe that cells derived from human embryonic stem cells (hESCs) in the grafts adopt mature dopaminergic signatures. We show that the presence of phenotypic dopaminergic genes, which were found to be differentially expressed in the transplants, is concentrated toward the edges of the grafts, in agreement with the immunohistochemical analyses. Deconvolution shows dopamine neurons being the dominating cell type in many features beneath the graft area. These findings further support the preferred environmental niche of TH-positive cells and confirm their dopaminergic phenotype through the presence of multiple dopaminergic markers.
ABSTRACT
Cell replacement therapies for Parkinson's disease (PD) based on transplantation of pluripotent stem cell-derived dopaminergic neurons are now entering clinical trials. Here, we present quality, safety, and efficacy data supporting the first-in-human STEM-PD phase I/IIa clinical trial along with the trial design. The STEM-PD product was manufactured under GMP and quality tested in vitro and in vivo to meet regulatory requirements. Importantly, no adverse effects were observed upon testing of the product in a 39-week rat GLP safety study for toxicity, tumorigenicity, and biodistribution, and a non-GLP efficacy study confirmed that the transplanted cells mediated full functional recovery in a pre-clinical rat model of PD. We further observed highly comparable efficacy results between two different GMP batches, verifying that the product can be serially manufactured. A fully in vivo-tested batch of STEM-PD is now being used in a clinical trial of 8 patients with moderate PD, initiated in 2022.
Subject(s)
Human Embryonic Stem Cells , Parkinson Disease , Humans , Rats , Animals , Parkinson Disease/therapy , Tissue Distribution , Cell Differentiation/physiology , Stem Cell Transplantation/methods , Dopaminergic Neurons/physiologyABSTRACT
The mucosal immune system identifies and fights invading pathogens, while allowing non-pathogenic organisms to persist. Mechanisms of pathogen/non-pathogen discrimination are poorly understood, as is the contribution of human genetic variation in disease susceptibility. We describe here a new, IRF3-dependent signaling pathway that is critical for distinguishing pathogens from normal flora at the mucosal barrier. Following uropathogenic E. coli infection, Irf3(-/-) mice showed a pathogen-specific increase in acute mortality, bacterial burden, abscess formation and renal damage compared to wild type mice. TLR4 signaling was initiated after ceramide release from glycosphingolipid receptors, through TRAM, CREB, Fos and Jun phosphorylation and p38 MAPK-dependent mechanisms, resulting in nuclear translocation of IRF3 and activation of IRF3/IFNß-dependent antibacterial effector mechanisms. This TLR4/IRF3 pathway of pathogen discrimination was activated by ceramide and by P-fimbriated E. coli, which use ceramide-anchored glycosphingolipid receptors. Relevance of this pathway for human disease was supported by polymorphic IRF3 promoter sequences, differing between children with severe, symptomatic kidney infection and children who were asymptomatic bacterial carriers. IRF3 promoter activity was reduced by the disease-associated genotype, consistent with the pathology in Irf3(-/-) mice. Host susceptibility to common infections like UTI may thus be strongly influenced by single gene modifications affecting the innate immune response.
Subject(s)
Immunity, Innate , Interferon Regulatory Factor-3/metabolism , Interferon Regulatory Factor-3/physiology , Kidney Neoplasms/etiology , Pyelonephritis/etiology , Signal Transduction , Urinary Tract Infections/etiology , Adult , Animals , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Case-Control Studies , Cell Nucleus/metabolism , Ceramides/metabolism , Child , Escherichia coli/pathogenicity , Escherichia coli Infections/etiology , Escherichia coli Infections/mortality , Escherichia coli Infections/prevention & control , Fimbriae, Bacterial , Gene Expression Profiling , Humans , Immunity, Innate/physiology , Interferon Regulatory Factor-3/genetics , Kidney/metabolism , Kidney/pathology , Kidney/virology , Kidney Neoplasms/mortality , Kidney Neoplasms/prevention & control , Lung Neoplasms/etiology , Lung Neoplasms/mortality , Lung Neoplasms/prevention & control , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Phosphorylation , Polymorphism, Genetic/genetics , Promoter Regions, Genetic/genetics , Prospective Studies , Protein Transport , Pyelonephritis/mortality , Pyelonephritis/pathology , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Tumor Cells, Cultured , Urinary Tract Infections/mortality , Urinary Tract Infections/prevention & controlABSTRACT
Human α-lactalbumin made lethal to tumor cells (HAMLET) is the first member in a new family of protein-lipid complexes that kills tumor cells with high selectivity. The protein component of HAMLET is α-lactalbumin, which in its native state acts as a substrate specifier in the lactose synthase complex, thereby defining a function essential for the survival of lactating mammals. In addition, α-lactalbumin acquires tumoricidal activity after partial unfolding and binding to oleic acid. The lipid cofactor serves the dual role as a stabilizer of the altered fold of the protein and a coactivator of specific steps in tumor cell death. HAMLET is broadly tumoricidal, suggesting that the complex identifies conserved death pathways suitable for targeting by novel therapies. Sensitivity to HAMLET is defined by oncogene expression including Ras and c-Myc and by glycolytic enzymes. Cellular targets are located in the cytoplasmic membrane, cytoskeleton, mitochondria, proteasomes, lysosomes and nuclei, and specific signaling pathways are rapidly activated, first by interactions of HAMLET with the cell membrane and subsequently after HAMLET internalization. Therapeutic effects of HAMLET have been demonstrated in human skin papillomas and bladder cancers, and HAMLET limits the progression of human glioblastomas, with no evidence of toxicity for normal brain or bladder tissue. These findings open up new avenues for cancer therapy and the understanding of conserved death responses in tumor cells.
Subject(s)
Glioblastoma , Lactalbumin/administration & dosage , Molecular Targeted Therapy , Oleic Acids/administration & dosage , Skin Neoplasms , Urinary Bladder Neoplasms , Cell Death/drug effects , Glioblastoma/drug therapy , Glioblastoma/metabolism , Humans , Lactalbumin/chemistry , Lactalbumin/metabolism , Lactose Synthase/chemistry , Lactose Synthase/metabolism , Oleic Acid/chemistry , Oleic Acid/metabolism , Oleic Acids/chemistry , Oleic Acids/metabolism , Signal Transduction , Skin Neoplasms/drug therapy , Skin Neoplasms/metabolism , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/metabolismABSTRACT
Human pluripotent stem cells (hPSCs) are intrinsically able to self-organize into cerebral organoids that mimic features of developing human brain tissue. These three-dimensional structures provide a unique opportunity to generate cytoarchitecture and cell-cell interactions reminiscent of human brain complexity in a dish. However, current in vitro brain organoid methodologies often result in intra-organoid variability, limiting their use in recapitulating later developmental stages as well as in disease modeling and drug discovery. In addition, cell stress and hypoxia resulting from long-term culture lead to incomplete maturation and cell death within the inner core. Here, we used a recombinant silk microfiber network as a scaffold to drive hPSCs to self-arrange into engineered cerebral organoids. Silk scaffolding promoted neuroectoderm formation and reduced heterogeneity of cellular organization within individual organoids. Bulk and single cell transcriptomics confirmed that silk cerebral organoids display more homogeneous and functionally mature neuronal properties than organoids grown in the absence of silk scaffold. Furthermore, oxygen sensing analysis showed that silk scaffolds create more favorable growth and differentiation conditions by facilitating the delivery of oxygen and nutrients. The silk scaffolding strategy appears to reduce intra-organoid variability and enhances self-organization into functionally mature human brain organoids.
ABSTRACT
We have developed an efficient approach to generate functional induced dopaminergic (DA) neurons from adult human dermal fibroblasts. When performing DA neuronal conversion of patient fibroblasts with idiopathic Parkinson's disease (PD), we could specifically detect disease-relevant pathology in these cells. We show that the patient-derived neurons maintain age-related properties of the donor and exhibit lower basal chaperone-mediated autophagy compared with healthy donors. Furthermore, stress-induced autophagy resulted in an age-dependent accumulation of macroautophagic structures. Finally, we show that these impairments in patient-derived DA neurons leads to an accumulation of phosphorylated alpha-synuclein, the classical hallmark of PD pathology. This pathological phenotype is absent in neurons generated from induced pluripotent stem cells from the same patients. Taken together, our results show that direct neural reprogramming can be used for obtaining patient-derived DA neurons, which uniquely function as a cellular model to study age-related pathology relevant to idiopathic PD.
Subject(s)
Induced Pluripotent Stem Cells , Parkinson Disease , Adult , Autophagy/physiology , Dopaminergic Neurons/pathology , Humans , Induced Pluripotent Stem Cells/pathology , Parkinson Disease/genetics , alpha-Synuclein/geneticsABSTRACT
Characterization of gene expression in pancreatic islets and its alteration in type 2 diabetes (T2D) are vital in understanding islet function and T2D pathogenesis. We leveraged RNA sequencing and genome-wide genotyping in islets from 188 donors to create the Islet Gene View (IGW) platform to make this information easily accessible to the scientific community. Expression data were related to islet phenotypes, diabetes status, other islet-expressed genes, islet hormone-encoding genes and for expression in insulin target tissues. The IGW web application produces output graphs for a particular gene of interest. In IGW, 284 differentially expressed genes (DEGs) were identified in T2D donor islets compared with controls. Forty percent of DEGs showed cell-type enrichment and a large proportion significantly co-expressed with islet hormone-encoding genes; glucagon (<i>GCG</i>, 56%), amylin (<i>IAPP</i>, 52%), insulin (<i>INS</i>, 44%), and somatostatin (<i>SST</i>, 24%). Inhibition of two DEGs, <i>UNC5D</i> and <i>SERPINE2</i>, impaired glucose-stimulated insulin secretion and impacted cell survival in a human ß-cell model. The exploratory use of IGW could help designing more comprehensive functional follow-up studies and serve to identify therapeutic targets in T2D.
Subject(s)
Diabetes Mellitus, Type 2 , Islets of Langerhans , Diabetes Mellitus, Type 2/genetics , Glucagon/genetics , Glucagon/metabolism , Humans , Insulin/genetics , Insulin/metabolism , Islets of Langerhans/metabolism , Serpin E2/metabolismABSTRACT
Human midbrain dopamine (DA) neurons are a heterogeneous group of cells that share a common neurotransmitter phenotype and are in close anatomical proximity but display different functions, sensitivity to degeneration, and axonal innervation targets. The A9 DA neuron subtype controls motor function and is primarily degenerated in Parkinson's disease (PD), whereas A10 neurons are largely unaffected by the condition, and their dysfunction is associated with neuropsychiatric disorders. Currently, DA neurons can only be reliably classified on the basis of topographical features, including anatomical location in the midbrain and projection targets in the forebrain. No systematic molecular classification at the genome-wide level has been proposed to date. Although many years of scientific efforts in embryonic and adult mouse brain have positioned us to better understand the complexity of DA neuron biology, many biological phenomena specific to humans are not amenable to being reproduced in animal models. The establishment of human cell-based systems combined with advanced computational single-cell transcriptomics holds great promise for decoding the mechanisms underlying maturation and diversification of human DA neurons, and linking their molecular heterogeneity to functions in the midbrain. Human pluripotent stem cells have emerged as a useful tool to recapitulate key molecular features of mature DA neuron subtypes. Here, we review some of the most recent advances and discuss the current challenges in using stem cells, to model human DA biology. We also describe how single cell RNA sequencing may provide key insights into the molecular programs driving DA progenitor specification into mature DA neuron subtypes. Exploiting the state-of-the-art approaches will lead to a better understanding of stem cell-derived DA neurons and their use in disease modeling and regenerative medicine.
Subject(s)
Dopaminergic Neurons/metabolism , Mesencephalon/metabolism , Parkinson Disease , Pluripotent Stem Cells/metabolism , RNA-Seq , Single-Cell Analysis , Animals , Dopaminergic Neurons/pathology , Humans , Mesencephalon/pathology , Parkinson Disease/genetics , Parkinson Disease/metabolism , Parkinson Disease/pathology , Pluripotent Stem Cells/pathologyABSTRACT
Dopaminergic (DA) neurons derived from human pluripotent stem cells (hPSCs) represent a renewable and available source of cells useful for understanding development, developing disease models, and stem-cell therapies for Parkinson's disease (PD). To assess the utility of stem cell cultures as an in vitro model system of human DA neurogenesis, we performed high-throughput transcriptional profiling of ~20,000 ventral midbrain (VM)-patterned stem cells at different stages of maturation using droplet-based single-cell RNA sequencing (scRNAseq). Using this dataset, we defined the cellular composition of human VM cultures at different timepoints and found high purity DA progenitor formation at an early stage of differentiation. DA neurons sharing similar molecular identities to those found in authentic DA neurons derived from human fetal VM were the major cell type after two months in culture. We also developed a bioinformatic pipeline that provided a comprehensive long noncoding RNA landscape based on temporal and cell-type specificity, which may contribute to unraveling the intricate regulatory network of coding and noncoding genes in DA neuron differentiation. Our findings serve as a valuable resource to elucidate the molecular steps of development, maturation, and function of human DA neurons, and to identify novel candidate coding and noncoding genes driving specification of progenitors into functionally mature DA neurons.