Publications by authors named "Vivi M Heine"

48 Publications

Neuron-Glia Interactions in Tuberous Sclerosis Complex Affect the Synaptic Balance in 2D and Organoid Cultures.

Cells 2021 Jan 12;10(1). Epub 2021 Jan 12.

Department of Child & Youth Psychiatry, Amsterdam UMC, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands.

Tuberous sclerosis complex (TSC) is a genetic disease affecting the brain. Neurological symptoms like epilepsy and neurodevelopmental issues cause a significant burden on patients. Both neurons and glial cells are affected by TSC mutations. Previous studies have shown changes in the excitation/inhibition balance (E/I balance) in TSC. Astrocytes are known to be important for neuronal development, and astrocytic dysfunction can cause changes in the E/I balance. We hypothesized that astrocytes affect the synaptic balance in TSC. TSC patient-derived stem cells were differentiated into astrocytes, which showed increased proliferation compared to control astrocytes. RNA sequencing revealed changes in gene expression, which were related to epidermal growth factor (EGF) signaling and enriched for genes that coded for secreted or transmembrane proteins. Control neurons were cultured in astrocyte-conditioned medium (ACM) of TSC and control astrocytes. After culture in TSC ACM, neurons showed an altered synaptic balance, with an increase in the percentage of VGAT synapses. These findings were confirmed in organoids, presenting a spontaneous 3D organization of neurons and glial cells. To conclude, this study shows that TSC astrocytes are affected and secrete factors that alter the synaptic balance. As an altered E/I balance may underlie many of the neurological TSC symptoms, astrocytes may provide new therapeutic targets.
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http://dx.doi.org/10.3390/cells10010134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826837PMC
January 2021

Evolution of adrenoleukodystrophy model systems.

J Inherit Metab Dis 2021 May 7;44(3):544-553. Epub 2021 Jan 7.

Department of Pediatric Neurology, Emma Children's Hospital, Amsterdam UMC, Amsterdam Leukodystrophy Center, Amsterdam Neuroscience, University of Amsterdam, Amsterdam, The Netherlands.

X-linked adrenoleukodystrophy (ALD) is a neurometabolic disorder affecting the adrenal glands, testes, spinal cord and brain. The disease is caused by mutations in the ABCD1 gene resulting in a defect in peroxisomal degradation of very long-chain fatty acids and their accumulation in plasma and tissues. Males with ALD have a near 100% life-time risk to develop myelopathy. The life-time prevalence to develop progressive cerebral white matter lesions (known as cerebral ALD) is about 60%. Adrenal insufficiency occurs in about 80% of male patients. In adulthood, 80% of women with ALD also develop myelopathy, but adrenal insufficiency or cerebral ALD are very rare. The complex clinical presentation and the absence of a genotype-phenotype correlation are complicating our understanding of the disease. In an attempt to understand the pathophysiology of ALD various model systems have been developed. While these model systems share the basic genetics and biochemistry of ALD they fail to fully recapitulate the complex neurodegenerative etiology of ALD. Each model system recapitulates certain aspects of the disorder. This exposes the complexity of ALD and therefore the challenge to create a comprehensive model system to fully understand ALD. In this review, we provide an overview of the different ALD modeling strategies from single-celled to multicellular organisms and from in vitro to in vivo approaches, and introduce how emerging iPSC-derived technologies could improve the understanding of this highly complex disorder.
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http://dx.doi.org/10.1002/jimd.12357DOI Listing
May 2021

Pharmacological intervention to restore connectivity deficits of neuronal networks derived from ASD patient iPSC with a TSC2 mutation.

Mol Autism 2020 10 19;11(1):80. Epub 2020 Oct 19.

Neuroscience and Mental Health Research Institute, Hadyn Ellis Building, Cathays, Cardiff, CF24 4HQ, UK.

Background: Tuberous sclerosis complex (TSC) is a rare genetic multisystemic disorder resulting from autosomal dominant mutations in the TSC1 or TSC2 genes. It is characterised by hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway and has severe neurodevelopmental and neurological components including autism, intellectual disability and epilepsy. In human and rodent models, loss of the TSC proteins causes neuronal hyperexcitability and synaptic dysfunction, although the consequences of these changes for the developing central nervous system are currently unclear.

Methods: Here we apply multi-electrode array-based assays to study the effects of TSC2 loss on neuronal network activity using autism spectrum disorder (ASD) patient-derived iPSCs. We examine both temporal synchronisation of neuronal bursting and spatial connectivity between electrodes across the network.

Results: We find that ASD patient-derived neurons with a functional loss of TSC2, in addition to possessing neuronal hyperactivity, develop a dysfunctional neuronal network with reduced synchronisation of neuronal bursting and lower spatial connectivity. These deficits of network function are associated with elevated expression of genes for inhibitory GABA signalling and glutamate signalling, indicating a potential abnormality of synaptic inhibitory-excitatory signalling. mTORC1 activity functions within a homeostatic triad of protein kinases, mTOR, AMP-dependent protein Kinase 1 (AMPK) and Unc-51 like Autophagy Activating Kinase 1 (ULK1) that orchestrate the interplay of anabolic cell growth and catabolic autophagy while balancing energy and nutrient homeostasis. The mTOR inhibitor rapamycin suppresses neuronal hyperactivity, but does not increase synchronised network activity, whereas activation of AMPK restores some aspects of network activity. In contrast, the ULK1 activator, LYN-1604, increases the network behaviour, shortens the network burst lengths and reduces the number of uncorrelated spikes.

Limitations: Although a robust and consistent phenotype is observed across multiple independent iPSC cultures, the results are based on one patient. There may be more subtle differences between patients with different TSC2 mutations or differences of polygenic background within their genomes. This may affect the severity of the network deficit or the pharmacological response between TSC2 patients.

Conclusions: Our observations suggest that there is a reduction in the network connectivity of the in vitro neuronal network associated with ASD patients with TSC2 mutation, which may arise via an excitatory/inhibitory imbalance due to increased GABA-signalling at inhibitory synapses. This abnormality can be effectively suppressed via activation of ULK1.
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http://dx.doi.org/10.1186/s13229-020-00391-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574213PMC
October 2020

Decanoic acid inhibits mTORC1 activity independent of glucose and insulin signaling.

Proc Natl Acad Sci U S A 2020 09 2;117(38):23617-23625. Epub 2020 Sep 2.

Centre for Biomedical Sciences, Department of Biological Sciences, Royal Holloway University of London, Egham TW20 0EX, United Kingdom;

Low-glucose and -insulin conditions, associated with ketogenic diets, can reduce the activity of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, potentially leading to a range of positive medical and health-related effects. Here, we determined whether mTORC1 signaling is also a target for decanoic acid, a key component of the medium-chain triglyceride (MCT) ketogenic diet. Using a tractable model system, , we show that decanoic acid can decrease mTORC1 activity, under conditions of constant glucose and in the absence of insulin, measured by phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). We determine that this effect of decanoic acid is dependent on a ubiquitin regulatory X domain-containing protein, mediating inhibition of a conserved AAA ATPase, p97, a homolog of the human transitional endoplasmic reticulum ATPase (VCP/p97) protein. We then demonstrate that decanoic acid decreases mTORC1 activity in the absence of insulin and under high-glucose conditions in ex vivo rat hippocampus and in tuberous sclerosis complex (TSC) patient-derived astrocytes. Our data therefore indicate that dietary decanoic acid may provide a new therapeutic approach to down-regulate mTORC1 signaling.
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http://dx.doi.org/10.1073/pnas.2008980117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519326PMC
September 2020

Glutamate Carrier Involvement in Mitochondrial Dysfunctioning in the Brain White Matter.

Front Mol Biosci 2020 21;7:151. Epub 2020 Jul 21.

Child and Youth Psychiatry, Emma Children's Hospital, Amsterdam UMC, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam, Netherlands.

Glutamate homeostasis is an important determinant of health of the central nervous system (CNS). Mitochondria play crucial roles in glutamate metabolism, especially in processes with a high energy demand such as action potential generation. Mitochondrial glutamate carriers (GCs) and aspartate-GCs (AGCs) regulate the transport of glutamate from the cytoplasm across the mitochondrial membrane, which is needed to control energy demand, lipid metabolism, and metabolic activity including oxidative phosphorylation and glycolysis. Dysfunction in these carriers are associated with seizures, spasticity, and/or myelin deficits, all of which are associated with inherited metabolic disorders. Since solute carrier functioning and associated processes are cell type- and context-specific, selective vulnerability to glutamate excitotoxicity and mitochondrial dysfunctioning is expected. Understanding this could offer important insights into the pathomechanisms of associated disorders. This perspective aims to explore the link between functions of both AGCs and GCs and their role in metabolic disorders, with a focus on a subclass of lysosomal storage disorders called leukodystrophies (LDs).
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http://dx.doi.org/10.3389/fmolb.2020.00151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385250PMC
July 2020

Cerebral Organoids: A Human Model for AAV Capsid Selection and Therapeutic Transgene Efficacy in the Brain.

Mol Ther Methods Clin Dev 2020 Sep 1;18:167-175. Epub 2020 Jun 1.

Department of Research & Development, uniQure Biopharma B.V., Amsterdam, the Netherlands.

The development of gene therapies for central nervous system disorders is challenging because it is difficult to translate preclinical data from current and models to the clinic. Therefore, we developed induced pluripotent stem cell (iPSC)-derived cerebral organoids as a model for recombinant adeno-associated virus (rAAV) capsid selection and for testing efficacy of AAV-based gene therapy in a human context. Cerebral organoids are physiological 3D structures that better recapitulate the human brain compared with 2D cell lines. To validate the model, we compared the transduction efficiency and distribution of two commonly used AAV serotypes (rAAV5 and rAAV9). In cerebral organoids, transduction with rAAV5 led to higher levels of vector DNA, transgenic mRNA, and protein expression as compared with rAAV9. The superior transduction of rAAV5 was replicated in iPSC-derived neuronal cells. Furthermore, rAAV5-mediated delivery of a human sequence-specific engineered microRNA to cerebral organoids led to a lower expression of its target ataxin-3. Our studies provide a new tool for selecting and deselecting AAV serotypes, and for demonstrating therapeutic efficacy of transgenes in a human context. Implementing cerebral organoids during gene therapy development could reduce the usage of animal models and improve translation to the clinic.
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http://dx.doi.org/10.1016/j.omtm.2020.05.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327852PMC
September 2020

Copy number variants (CNVs): a powerful tool for iPSC-based modelling of ASD.

Mol Autism 2020 06 1;11(1):42. Epub 2020 Jun 1.

Neuroscience & Mental Health Research Institute, Cardiff University, Cardiff, CF24 4HQ, UK.

Patients diagnosed with chromosome microdeletions or duplications, known as copy number variants (CNVs), present a unique opportunity to investigate the relationship between patient genotype and cell phenotype. CNVs have high genetic penetrance and give a good correlation between gene locus and patient clinical phenotype. This is especially effective for the study of patients with neurodevelopmental disorders (NDD), including those falling within the autism spectrum disorders (ASD). A key question is whether this correlation between genetics and clinical presentation at the level of the patient can be translated to the cell phenotypes arising from the neurodevelopment of patient induced pluripotent stem cells (iPSCs).Here, we examine how iPSCs derived from ASD patients with an associated CNV inform our understanding of the genetic and biological mechanisms underlying the aetiology of ASD. We consider selection of genetically characterised patient iPSCs; use of appropriate control lines; aspects of human neurocellular biology that can capture in vitro the patient clinical phenotype; and current limitations of patient iPSC-based studies. Finally, we consider how future research may be enhanced to maximise the utility of CNV patients for research of pathological mechanisms or therapeutic targets.
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http://dx.doi.org/10.1186/s13229-020-00343-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7268297PMC
June 2020

Quantitative proteomic analysis of Rett iPSC-derived neuronal progenitors.

Mol Autism 2020 05 27;11(1):38. Epub 2020 May 27.

Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.

Background: Rett syndrome (RTT) is a progressive neurodevelopmental disease that is characterized by abnormalities in cognitive, social, and motor skills. RTT is often caused by mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). The mechanism by which impaired MeCP2 induces the pathological abnormalities in the brain is not understood. Both patients and mouse models have shown abnormalities at molecular and cellular level before typical RTT-associated symptoms appear. This implies that underlying mechanisms are already affected during neurodevelopmental stages.

Methods: To understand the molecular mechanisms involved in disease onset, we used an RTT patient induced pluripotent stem cell (iPSC)-based model with isogenic controls and performed time-series of proteomic analysis using in-depth high-resolution quantitative mass spectrometry during early stages of neuronal development.

Results: We provide mass spectrometry-based quantitative proteomic data, depth of about 7000 proteins, at neuronal progenitor developmental stages of RTT patient cells and isogenic controls. Our data gives evidence of proteomic alteration at early neurodevelopmental stages, suggesting alterations long before the phase that symptoms of RTT syndrome become apparent. Significant changes are associated with the GO enrichment analysis in biological processes cell-cell adhesion, actin cytoskeleton organization, neuronal stem cell population maintenance, and pituitary gland development, next to protein changes previously associated with RTT, i.e., dendrite morphology and synaptic deficits. Differential expression increased from early to late neural stem cell phases, although proteins involved in immunity, metabolic processes, and calcium signaling were affected throughout all stages analyzed.

Limitations: The limitation of our study is the number of RTT patients analyzed. As the aim of our study was to investigate a large number of proteins, only one patient was considered, of which 3 different RTT iPSC clones and 3 isogenic control iPSC clones were included. Even though this approach allowed the study of mutation-induced alterations due to the usage of isogenic controls, results should be validated on different RTT patients to suggest common disease mechanisms.

Conclusions: During early neuronal differentiation, there are consistent and time-point specific proteomic alterations in RTT patient cells carrying exons 3-4 deletion in MECP2. We found changes in proteins involved in pathway associated with RTT phenotypes, including dendrite morphology and synaptogenesis. Our results provide a valuable resource of proteins and pathways for follow-up studies, investigating common mechanisms involved during early disease stages of RTT syndrome.
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http://dx.doi.org/10.1186/s13229-020-00344-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251722PMC
May 2020

KCC2 expression levels are reduced in post mortem brain tissue of Rett syndrome patients.

Acta Neuropathol Commun 2019 12 3;7(1):196. Epub 2019 Dec 3.

Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit, Amsterdam, The Netherlands.

Rett Syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the Methyl CpG binding protein 2 (MECP2) gene. Deficient K-Clco-transporter 2 (KCC2) expression is suggested to play a key role in the neurodevelopmental delay in RTT patients' neuronal networks. KCC2 is a major player in neuronal maturation by supporting the GABAergic switch, through the regulation of neuronal chlorine homeostasis. Previous studies suggest that MeCP2 mutations lead to changed KCC2 expression levels, thereby causing a disturbance in excitation/inhibition (E/I) balance. To investigate this, we performed protein and RNA expression analysis on post mortem brain tissue from RTT patients and healthy controls. We showed that KCC2 expression, in particular the KCC2a isoform, is relatively decreased in RTT patients. The expression of Na-K-Cl co-transporter 1 (NKCC1), responsible for the inward transport of chlorine, is not affected, leading to a reduced KCC2/NKCC1 ratio in RTT brains. Our report confirms KCC2 expression alterations in RTT patients in human brain tissue, which is in line with other studies, suggesting affected E/I balance could underlie neurodevelopmental defects in RTT patients.
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http://dx.doi.org/10.1186/s40478-019-0852-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892240PMC
December 2019

Co-culture of Human Stem Cell Derived Neurons and Oligodendrocyte Progenitor Cells.

Bio Protoc 2019 Sep 5;9(17):e3350. Epub 2019 Sep 5.

Pediatric Neurology, Emma Children's Hospital, Amsterdam UMC, Amsterdam, Neuroscience, Vrije Universiteit Amsterdam, The Netherlands.

Crosstalk between neurons and oligodendrocytes is important for proper brain functioning. Multiple co-culture methods have been developed to study oligodendrocyte maturation, myelination or the effect of oligodendrocytes on neurons. However, most of these methods contain cells derived from animal models. In the current protocol, we co-culture human neurons with human oligodendrocytes. Neurons and oligodendrocyte precursor cells (OPCs) were differentiated separately from pluripotent stem cells according to previously published protocols. To study neuron-glia cross-talk, neurons and OPCs were plated in co-culture mode in optimized conditions for additional 28 days, and prepared for OPC maturation and neuronal morphology analysis. To our knowledge, this is one of the first neuron-OPC protocols containing all human cells. Specific neuronal abnormalities not observed in mono-cultures of Tuberous Sclerosis Complex (TSC) neurons, became apparent when TSC neurons were co-cultured with TSC OPCs. These results show that this co-culture system can be used to study human neuron-OPC interactive mechanisms involved in health and disease.
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http://dx.doi.org/10.21769/BioProtoc.3350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854112PMC
September 2019

Astrocyte Subtype Vulnerability in Stem Cell Models of Vanishing White Matter.

Ann Neurol 2019 11 10;86(5):780-792. Epub 2019 Sep 10.

Department of Pediatric Neurology, Emma Children's Hospital, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam Neuroscience, Amsterdam, the Netherlands.

Objective: Astrocytes have gained attention as important players in neurological disease. In line with their heterogeneous character, defects in specific astrocyte subtypes have been identified. Leukodystrophy vanishing white matter (VWM) shows selective vulnerability in white matter astrocytes, but the underlying mechanisms remain unclear. Induced pluripotent stem cell technology is being extensively explored in studies of pathophysiology and regenerative medicine. However, models for distinct astrocyte subtypes for VWM are lacking, thereby hampering identification of disease-specific pathways.

Methods: Here, we characterize human and mouse pluripotent stem cell-derived gray and white matter astrocyte subtypes to generate an in vitro VWM model. We examined morphology and functionality, and used coculture methods, high-content microscopy, and RNA sequencing to study VWM cultures.

Results: We found intrinsic vulnerability in specific astrocyte subpopulations in VWM. When comparing VWM and control cultures, white matter-like astrocytes inhibited oligodendrocyte maturation, and showed affected pathways in both human and mouse cultures, involving the immune system and extracellular matrix. Interestingly, human white matter-like astrocytes presented additional, human-specific disease mechanisms, such as neuronal and mitochondrial functioning.

Interpretation: Astrocyte subtype cultures revealed disease-specific pathways in VWM. Cross-validation of human- and mouse-derived protocols identified human-specific disease aspects. This study provides new insights into VWM disease mechanisms, which helps the development of in vivo regenerative applications, and we further present strategies to study astrocyte subtype vulnerability in neurological disease. ANN NEUROL 2019;86:780-792.
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http://dx.doi.org/10.1002/ana.25585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856690PMC
November 2019

The involvement of astrocytes in early-life adversity induced programming of the brain.

Glia 2019 09 30;67(9):1637-1653. Epub 2019 Apr 30.

Center for Neuroscience, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.

Early-life adversity (ELA) in the form of stress, inflammation, or malnutrition, can increase the risk of developing psychopathology or cognitive problems in adulthood. The neurobiological substrates underlying this process remain unclear. While neuronal dysfunction and microglial contribution have been studied in this context, only recently the role of astrocytes in early-life programming of the brain has been appreciated. Astrocytes serve many basic roles for brain functioning (e.g., synaptogenesis, glutamate recycling), and are unique in their capacity of sensing and integrating environmental signals, as they are the first cells to encounter signals from the blood, including hormonal changes (e.g., glucocorticoids), immune signals, and nutritional information. Integration of these signals is especially important during early development, and therefore we propose that astrocytes contribute to ELA induced changes in the brain by sensing and integrating environmental signals and by modulating neuronal development and function. Studies in rodents have already shown that ELA can impact astrocytes on the short and long term, however, a critical review of these results is currently lacking. Here, we will discuss the developmental trajectory of astrocytes, their ability to integrate stress, immune, and nutritional signals from the early environment, and we will review how different types of early adversity impact astrocytes.
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http://dx.doi.org/10.1002/glia.23625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6767561PMC
September 2019

Cell Replacement Therapy Improves Pathological Hallmarks in a Mouse Model of Leukodystrophy Vanishing White Matter.

Stem Cell Reports 2019 03 21;12(3):441-450. Epub 2019 Feb 21.

Pediatric Neurology, Emma Children's Hospital, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam Neuroscience, 1081 HV Amsterdam, the Netherlands; Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, the Netherlands. Electronic address:

Stem cell therapy has great prospects for brain white matter disorders, including the genetically determined disorders called leukodystrophies. We focus on the devastating leukodystrophy vanishing white matter (VWM). Patients with VWM show severe disability and early death, and treatment options are lacking. Previous studies showed successful cell replacement therapy in rodent models for myelin defects. However, proof-of-concept studies of allogeneic cell replacement in models representative of human leukodystrophies are lacking. We tested cell replacement in a mouse model representative of VWM. We transplanted different murine glial progenitor cell populations and showed improved pathological hallmarks and motor function. Improved mice showed a higher percentage of transplanted cells that differentiated into GFAP astrocytes, suggesting best therapeutic prospects for replacement of astroglial lineage cells. This is a proof-of-concept study for cell transplantation in VWM and suggests that glial cell replacement therapy is a promising therapeutic strategy for leukodystrophy patients.
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http://dx.doi.org/10.1016/j.stemcr.2019.01.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411482PMC
March 2019

Neuron-Glia Interactions Increase Neuronal Phenotypes in Tuberous Sclerosis Complex Patient iPSC-Derived Models.

Stem Cell Reports 2019 01 20;12(1):42-56. Epub 2018 Dec 20.

Pediatric Neurology, Emma Children's Hospital, Amsterdam UMC, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam 1081 HV, the Netherlands; Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam 1081 HV, the Netherlands. Electronic address:

Tuberous sclerosis complex (TSC) is a rare neurodevelopmental disorder resulting from autosomal dominant mutations in the TSC1 or TSC2 genes, leading to a hyperactivated mammalian target of rapamycin (mTOR) pathway, and gray and white matter defects in the brain. To study the involvement of neuron-glia interactions in TSC phenotypes, we generated TSC patient induced pluripotent stem cell (iPSC)-derived cortical neuronal and oligodendrocyte (OL) cultures. TSC neuron mono-cultures showed increased network activity, as measured by calcium transients and action potential firing, and increased dendritic branching. However, in co-cultures with OLs, neuronal defects became more apparent, showing cellular hypertrophy and increased axonal density. In addition, TSC neuron-OL co-cultures showed increased OL cell proliferation and decreased OL maturation. Pharmacological intervention with the mTOR regulator rapamycin suppressed these defects. Our patient iPSC-based model, therefore, shows a complex cellular TSC phenotype arising from the interaction of neuronal and glial cells and provides a platform for TSC disease modeling and drug development.
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http://dx.doi.org/10.1016/j.stemcr.2018.11.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335594PMC
January 2019

Generation of Isogenic Controls for In Vitro Disease Modelling of X-Chromosomal Disorders.

Stem Cell Rev Rep 2019 04;15(2):276-285

Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Boelelaan 1085, 1081HV, Amsterdam, The Netherlands.

Generation of proper controls is crucial in induced pluripotent stem cell (iPSC) studies. X-chromosomal disorders offer the potential to develop isogenic controls due to random X-chromosomal inactivation (XCI). However, the generation of such lines is currently hampered by skewed X-inactivation in fibroblast lines and X-chromosomal reactivation (XCR) after reprogramming. Here we describe a method to generate a pure iPSC population with respect to the specific inactivated X-chromosome (Xi). We used fibroblasts from Rett patients, who all have a causal mutation in the X-linked MeCP2 gene. Pre-sorting these fibroblasts followed by episomal reprogramming, allowed us to overcome skewness in fibroblast lines and to retain the X-chromosomal state, which was unpredictable with lentiviral reprogramming. This means that fibroblast pre-sorting followed by episomal reprogramming can be used to reliably generate iPSC lines with specified X-chromosomal phenotype such as Rett syndrome.
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http://dx.doi.org/10.1007/s12015-018-9851-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441401PMC
April 2019

Patterning factors during neural progenitor induction determine regional identity and differentiation potential in vitro.

Stem Cell Res 2018 10 23;32:25-34. Epub 2018 Aug 23.

Pediatric Neurology, Emma Children's Hospital, Amsterdam UMC, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, the Netherlands; Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, the Netherlands. Electronic address:

The neural tube consists of neural progenitors (NPs) that acquire different characteristics during gestation due to patterning factors. However, the influence of such patterning factors on human pluripotent stem cells (hPSCs) during in vitro neural differentiation is often unclear. This study compared neural induction protocols involving in vitro patterning with single SMAD inhibition (SSI), retinoic acid (RA) administration and dual SMAD inhibition (DSI). While the derived NP cells expressed known NP markers, they differed in their NP expression profile and differentiation potential. Cortical neuronal cells generated from 1) SSI NPs exhibited less mature neuronal phenotypes, 2) RA NPs exhibited an increased GABAergic phenotype, and 3) DSI NPs exhibited greater expression of glutamatergic lineage markers. Further, although all NPs generated astrocytes, astrocytes derived from the RA-induced NPs had the highest GFAP expression. Differences between NP populations included differential expression of regional identity markers HOXB4, LBX1, OTX1 and GSX2, which persisted into mature neural cell stages. This study suggests that patterning factors regulate how potential NPs may differentiate into specific neuronal and glial cell types in vitro. This challenges the utility of generic neural induction procedures, while highlighting the importance of carefully selecting specific NP protocols.
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http://dx.doi.org/10.1016/j.scr.2018.08.017DOI Listing
October 2018

Axonal abnormalities in vanishing white matter.

Ann Clin Transl Neurol 2018 Apr 1;5(4):429-444. Epub 2018 Mar 1.

Department of Pediatrics/Child Neurology Amsterdam Neuroscience VU University Medical Centre Amsterdam The Netherlands.

Objective: We aimed to study the occurrence and development of axonal pathology and the influence of astrocytes in vanishing white matter.

Methods: Axons and myelin were analyzed using electron microscopy and immunohistochemistry on and single- and double-mutant mice and patient brain tissue. In addition, astrocyte-forebrain co-culture studies were performed.

Results: In the corpus callosum of mutant mice, myelin sheath thickness, axonal diameter, and G-ratio developed normally up to 4 months. At 7 months, however, axons had become thinner, while in control mice axonal diameters had increased further. Myelin sheath thickness remained close to normal, resulting in an abnormally low G-ratio in mutant mice. In more severely affected double-mutants, similar abnormalities were already present at 4 months, while in milder affected mutants, few abnormalities were observed at 7 months. Additionally, from 2 months onward an increased percentage of thin, unmyelinated axons and increased axonal density were present in -mutant mice. Co-cultures showed that mutant astrocytes induced increased axonal density, also in control forebrain tissue, and that control astrocytes induced normal axonal density, also in mutant forebrain tissue. In vanishing white matter patient brains, axons and myelin sheaths were thinner than normal in moderately and severely affected white matter. In mutant mice and patients, signs of axonal transport defects and cytoskeletal abnormalities were minimal.

Interpretation: In vanishing white matter, axons are initially normal and atrophy later. Astrocytes are central in this process. If therapy becomes available, axonal pathology may be prevented with early intervention.
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http://dx.doi.org/10.1002/acn3.540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899913PMC
April 2018

Adult mouse eIF2Bε Arg191His astrocytes display a normal integrated stress response in vitro.

Sci Rep 2018 02 28;8(1):3773. Epub 2018 Feb 28.

Department of Pediatrics/Child Neurology, VU University Medical Center, Amsterdam, The Netherlands.

Vanishing white matter (VWM) is a genetic childhood white matter disorder, characterized by chronic as well as episodic, stress provoked, neurological deterioration. Treatment is unavailable and patients often die within a few years after onset. VWM is caused by recessive mutations in the eukaryotic initiation factor 2B (eIF2B). eIF2B regulates protein synthesis rates in every cell of the body. In normal cells, various types of cellular stress inhibit eIF2B activity and induce the integrated stress response (ISR). We have developed a VWM mouse model homozygous for the pathogenic Arg191His mutation in eIF2Bε (2b5 ), representative of the human disease. Neuropathological examination of VWM patient and mouse brain tissue suggests that astrocytes are primarily affected. We hypothesized that VWM astrocytes are selectively hypersensitive to ISR induction, resulting in a heightened response. We cultured astrocytes from wildtype and VWM mice and investigated the ISR in assays that measure transcriptional induction of stress genes, protein synthesis rates and cell viability. We investigated the effects of short- and long-term stress as well as stress recovery. We detected congruent results amongst the various assays and did not detect a hyperactive ISR in VWM mouse astrocytes.
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http://dx.doi.org/10.1038/s41598-018-21885-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830650PMC
February 2018

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification.

J Vis Exp 2017 12 9(130). Epub 2017 Dec 9.

Department of Pediatrics/Child Neurology, Amsterdam Neuroscience, VU University Medical Center; Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam;

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended effects that extrinsic patterning factors might introduce into human pluripotent stem cell (hPSC) models of brain development or pathophysiology, such as masking disease phenotype. Here, we present two cerebellar differentiation protocols for hPSCs, designed with simpler startup method, fewer patterning factors, and less material requirements than previous protocols. Recently, we developed culture procedures, which generate free-floating 3-dimensional (3D) products consistent with other brain "organoid" protocols, including morphologies relevant to modeling brain development such as sub/ventricular zone- and rhombic lip-like structures. The second uses an adherent, 2D monolayer procedure to complete differentiation, which is shown capable of generating functional cerebellar neurons, as products are positive for cerebellar-associated markers, and exhibit neuron-like calcium influxes. Together, these protocols offer scientists a choice of options suited to different research purposes, as well as a basic model for testing other types of streamlined neural differentiations.
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http://dx.doi.org/10.3791/56888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5755539PMC
December 2017

Affected astrocytes in the spinal cord of the leukodystrophy vanishing white matter.

Glia 2018 04 29;66(4):862-873. Epub 2017 Dec 29.

Department of Pediatrics/Child Neurology, Amsterdam Neuroscience, VU University Medical Center, Amsterdam, The Netherlands.

Leukodystrophies are often devastating diseases, presented with progressive clinical signs as spasticity, ataxia and cognitive decline, and lack proper treatment options. New therapy strategies for leukodystrophies mostly focus on oligodendrocyte replacement to rescue lack of myelin in the brain, even though disease pathology also often involves other glial cells and the spinal cord. In this study we investigated spinal cord pathology in a mouse model for Vanishing White Matter disease (VWM) and show that astrocytes in the white matter are severely affected. Astrocyte pathology starts postnatally in the sensory tracts, followed by changes in the astrocytic populations in the motor tracts. Studies in post-mortem tissue of two VWM patients, a 13-year-old boy and a 6-year-old girl, confirmed astrocyte abnormalities in the spinal cord. For proper development of new treatment options for VWM and, possibly, other leukodystrophies, future studies should investigate spinal cord involvement.
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http://dx.doi.org/10.1002/glia.23289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5838785PMC
April 2018

The Healthy and Diseased Microenvironments Regulate Oligodendrocyte Properties: Implications for Regenerative Medicine.

Am J Pathol 2018 01 10;188(1):39-52. Epub 2017 Oct 10.

Department of Pediatrics/Child Neurology, VU University Medical Center, Amsterdam Neuroscience, Amsterdam, the Netherlands; Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands. Electronic address:

White matter disorders are characterized by deficient myelin or myelin loss, lead to a range of neurologic dysfunctions, and can result in early death. Oligodendrocytes, which are responsible for white matter formation, are the first targets for treatment. However, many studies indicate that failure of white matter repair goes beyond the intrinsic incapacity of oligodendrocytes to (re)generate myelin and that failed interactions with neighboring cells or factors in the diseased microenvironment can underlie white matter defects. Moreover, most of the white matter disorders show specific white matter pathology caused by different disease mechanisms. Herein, we review the factors within the cellular and the extracellular microenvironment regulating oligodendrocyte properties and discuss stem cell tools to identify microenvironmental factors of importance to the development of improved regenerative medicine for patients with white matter disorders.
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http://dx.doi.org/10.1016/j.ajpath.2017.08.030DOI Listing
January 2018

Comparative gene expression study and pathway analysis of the human iris- and the retinal pigment epithelium.

PLoS One 2017 21;12(8):e0182983. Epub 2017 Aug 21.

Department of Clinical Genetics, Academic Medical Center, Amsterdam, The Netherlands.

Background: The retinal pigment epithelium (RPE) is a neural monolayer lining the back of the eye. Degeneration of the RPE leads to severe vision loss in, so far incurable, diseases such as age-related macular degeneration and some forms of retinitis pigmentosa. A promising future replacement therapy may be autologous iris epithelial cell transdifferentiation into RPE in vitro and, subsequently, transplantation. In this study we compared the gene expression profiles of the iris epithelium (IE) and the RPE.

Methods: We collected both primary RPE- and IE cells from 5 freshly frozen human donor eyes, using respectively laser dissection microscopy and excision. We performed whole-genome expression profiling using 44k Agilent human microarrays. We investigated the gene expression profiles on both gene and functional network level, using R and the knowledge database Ingenuity.

Results: The major molecular pathways related to the RPE and IE were quite similar and yielded basic neuro-epithelial cell functions. Nonetheless, we also found major specific differences: For example, genes and molecular pathways, related to the visual cycle and retinol biosynthesis are significantly higher expressed in the RPE than in the IE. Interestingly, Wnt and aryl hydrocarbon receptor (AhR-) signaling pathways are much higher expressed in the IE than in the RPE, suggesting, respectively, a possible pluripotent and high detoxification state of the IE.

Conclusions: This study provides a valuation of the similarities and differences between the expression profiles of the RPE and IE. Our data combined with that of the literature, represent a most comprehensive perspective on transcriptional variation, which may support future research in the development of therapeutic transplantation of IE.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0182983PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5565104PMC
October 2017

Genetically-Informed Patient Selection for iPSC Studies of Complex Diseases May Aid in Reducing Cellular Heterogeneity.

Front Cell Neurosci 2017 13;11:164. Epub 2017 Jun 13.

Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Vrije Universiteit AmsterdamAmsterdam, Netherlands.

Induced pluripotent stem cell (iPSC) technology is more and more used for the study of genetically complex human disease but is challenged by variability, sample size and polygenicity. We discuss studies involving iPSC-derived neurons from patients with Schizophrenia (SCZ), to exemplify that heterogeneity in sampling strategy complicate the detection of disease mechanisms. We offer a solution to controlling variability within and between iPSC studies by using specific patient selection strategies.
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http://dx.doi.org/10.3389/fncel.2017.00164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5468546PMC
June 2017

Multi-level characterization of balanced inhibitory-excitatory cortical neuron network derived from human pluripotent stem cells.

PLoS One 2017 6;12(6):e0178533. Epub 2017 Jun 6.

Department of Pediatrics / Child Neurology, Amsterdam Neuroscience, VU University Medical Center, Amsterdam, The Netherlands.

Generation of neuronal cultures from induced pluripotent stem cells (hiPSCs) serve the studies of human brain disorders. However we lack neuronal networks with balanced excitatory-inhibitory activities, which are suitable for single cell analysis. We generated low-density networks of hPSC-derived GABAergic and glutamatergic cortical neurons. We used two different co-culture models with astrocytes. We show that these cultures have balanced excitatory-inhibitory synaptic identities using confocal microscopy, electrophysiological recordings, calcium imaging and mRNA analysis. These simple and robust protocols offer the opportunity for single-cell to multi-level analysis of patient hiPSC-derived cortical excitatory-inhibitory networks; thereby creating advanced tools to study disease mechanisms underlying neurodevelopmental disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178533PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460818PMC
September 2017

Differential Maturation of the Two Regulated Secretory Pathways in Human iPSC-Derived Neurons.

Stem Cell Reports 2017 03 23;8(3):659-672. Epub 2017 Feb 23.

Departments of Functional Genomics and Clinical Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit (VU) Amsterdam and VU Medical Center, de Boelelaan 1087, 1081 HV Amsterdam, the Netherlands. Electronic address:

Neurons communicate by regulated secretion of chemical signals from synaptic vesicles (SVs) and dense-core vesicles (DCVs). Here, we investigated the maturation of these two secretory pathways in micro-networks of human iPSC-derived neurons. These micro-networks abundantly expressed endogenous SV and DCV markers, including neuropeptides. DCV transport was microtubule dependent, preferentially anterograde in axons, and 2-fold faster in axons than in dendrites. SV and DCV secretion were strictly Ca and SNARE dependent. DCV secretion capacity matured until day in vitro (DIV) 36, with intense stimulation releasing 6% of the total DCV pool, and then plateaued. This efficiency is comparable with mature mouse neurons. In contrast, SV secretion capacity continued to increase until DIV50, with substantial further increase in secretion efficiency and decrease in silent synapses. These data show that the two secretory pathways can be studied in human neurons and that they mature differentially, with DCV secretion reaching maximum efficiency when that of SVs is still low.
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http://dx.doi.org/10.1016/j.stemcr.2017.01.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355645PMC
March 2017

Simplified 3D protocol capable of generating early cortical neuroepithelium.

Biol Open 2017 Mar 15;6(3):402-406. Epub 2017 Mar 15.

Department of Pediatrics/Child Neurology, Amsterdam Neuroscience, VU University Medical Center, Amsterdam 1081 HV, The Netherlands

Here, we report a 3D cerebellar differentiation protocol with quick startup method, defined medium and no special materials or handling requirements. Three fibroblast growth factors (FGF2, 4 and 8) were used for cerebellar patterning and smoothened agonist (SAG) for granule cell development. After 35 days, differentiation products exhibited similar structures and neuronal markers reported in prior 'organoid' and 'spheroid' protocols. This included cells positive for KIRREL2 (a marker of early cerebellar neuroepithelium) and ZIC1 (a marker for granule cells). Follow-up tests indicated that addition of FGFs, if helpful, was not required to generate observed structures and cell types. This suggests that intrinsic production of patterning factors by aggregates themselves may be adequate for region-specific 3D modeling. This protocol may be used as a quick, easy and cost-efficient method for 3D culture, whether to research development of the early cerebellar neuroepithelium, a base to generate mature cortical structures, or to optimize minimal-factor protocols for other brain regions.
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http://dx.doi.org/10.1242/bio.021725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5374398PMC
March 2017

Leukodystrophies: Five new things.

Neurol Clin Pract 2016 Dec;6(6):506-514

Department of Child Neurology (MSvdK, NIW, VMH), Amsterdam Neuroscience, VU University Medical Centre, Amsterdam; and Departments of Functional Genomics (MSvdK) and Complex Trait Genetics (VMH), Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University, Amsterdam, the Netherlands.

Purpose Of Review: Leukodystrophies are genetic disorders primarily and predominantly affecting CNS white matter. They are associated with connotations such as "much unknown," "progressive myelin loss," and "nothing can be done." Recent technological progress is reversing this picture.

Recent Findings: Next-generation sequencing has created the revolution of whole-exome/genome sequencing, allowing disease definition and gene identification for numerous ultra-rare disorders by focusing on very small groups and individual patients. Knowledge of many new "white matter proteins" is transforming our understanding of white matter physiology and pathophysiology. Regarding therapy, especially stem cell and gene therapy are evolving rapidly, aiming at personalized therapy for a specific patient with a specific disease. Multimodal approaches targeting multiple aspects of the disease hold the highest promise.

Summary: Technological developments are revolutionizing the leukodystrophy field. Unknown becomes known and untreatable becomes treatable. New insight is that not all leukodystrophies are irreversible and that some improve spontaneously.
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http://dx.doi.org/10.1212/CPJ.0000000000000289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964825PMC
December 2016

Stem cell therapy for white matter disorders: don't forget the microenvironment!

J Inherit Metab Dis 2016 07 21;39(4):513-8. Epub 2016 Mar 21.

Department of Pediatrics/Child Neurology, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.

White matter disorders (WMDs) are a major source of handicap at all ages. They often lead to progressive neurological dysfunction and early death. Although causes are highly diverse, WMDs share the property that glia (astrocytes and oligodendrocytes) are among the cells primarily affected, and that myelin is either not formed or lost. Many WMDs might benefit from cell replacement therapies. Successful preclinical studies in rodent models have already led to the first clinical trials in humans using glial or oligodendrocyte progenitor cells aiming at (re)myelination. However, myelin is usually not the only affected structure. Neurons, microglia, and astrocytes are often also affected and are all important partners in creating the right conditions for proper white matter repair. Composition of the extracellular environment is another factor to be considered. Cell transplantation therapies might therefore require inclusion of non-oligodendroglial cell types and target more than only myelin repair. WMD patients would likely benefit from multimodal therapy approaches involving stem cell transplantation and microenvironment-targeting strategies to alter the local environment to a more favorable state for cell replacement. Furthermore most proof-of-concept studies have been performed with human cells in rodent disease models. Since human glial cells show a larger regenerative capacity than their mouse counterparts in the host mouse brain, microenvironmental factors affecting white matter recovery might be overlooked in rodent studies. We would like to stress that cell replacement therapy is a highly promising therapeutic option for WMDs, but a receptive microenvironment is crucial.
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http://dx.doi.org/10.1007/s10545-016-9925-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920834PMC
July 2016

Astrocytes are central in the pathomechanisms of vanishing white matter.

J Clin Invest 2016 Apr 14;126(4):1512-24. Epub 2016 Mar 14.

Vanishing white matter (VWM) is a fatal leukodystrophy that is caused by mutations in genes encoding subunits of eukaryotic translation initiation factor 2B (eIF2B). Disease onset and severity are codetermined by genotype. White matter astrocytes and oligodendrocytes are almost exclusively affected; however, the mechanisms of VWM development remain unclear. Here, we used VWM mouse models, patients' tissue, and cell cultures to investigate whether astrocytes or oligodendrocytes are the primary affected cell type. We generated 2 mouse models with mutations (Eif2b5Arg191His/Arg191His and Eif2b4Arg484Trp/Arg484Trp) that cause severe VWM in humans and then crossed these strains to develop mice with various mutation combinations. Phenotypic severity was highly variable and dependent on genotype, reproducing the clinical spectrum of human VWM. In all mutant strains, impaired maturation of white matter astrocytes preceded onset and paralleled disease severity and progression. Bergmann glia and retinal Müller cells, nonforebrain astrocytes that have not been associated with VWM, were also affected, and involvement of these cells was confirmed in VWM patients. In coculture, VWM astrocytes secreted factors that inhibited oligodendrocyte maturation, whereas WT astrocytes allowed normal maturation of VWM oligodendrocytes. These studies demonstrate that astrocytes are central in VWM pathomechanisms and constitute potential therapeutic targets. Importantly, astrocytes should also be considered in the pathophysiology of other white matter disorders.
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http://dx.doi.org/10.1172/JCI83908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4811153PMC
April 2016

Comparison of Mouse and Human Retinal Pigment Epithelium Gene Expression Profiles: Potential Implications for Age-Related Macular Degeneration.

PLoS One 2015 30;10(10):e0141597. Epub 2015 Oct 30.

Department of Clinical Genetics, Academic Medical Centre, Amsterdam, The Netherlands; The Netherlands Institute for Neuroscience (NIN-KNAW), Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands; Department of Ophthalmology, Academic Medical Centre, Amsterdam, The Netherlands.

Background: The human retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age related macular degeneration (AMD). AMD is the leading cause of blindness worldwide. There is currently no effective treatment available. Preclinical studies in AMD mouse models are essential to develop new therapeutics. This requires further in-depth knowledge of the similarities and differences between mouse and human RPE.

Methods: We performed a microarray study to identify and functionally annotate RPE specific gene expression in mouse and human RPE. We used a meticulous method to determine C57BL/6J mouse RPE signature genes, correcting for possible RNA contamination from its adjacent layers: the choroid and the photoreceptors. We compared the signature genes, gene expression profiles and functional annotations of the mouse and human RPE.

Results: We defined sets of mouse (64), human (171) and mouse-human interspecies (22) RPE signature genes. Not unexpectedly, our gene expression analysis and comparative functional annotation suggested that, in general, the mouse and human RPE are very similar. For example, we found similarities for general features, like "organ development" and "disorders related to neurological tissue". However, detailed analysis of the molecular pathways and networks associated with RPE functions, suggested also multiple species-specific differences, some of which may be relevant for the development of AMD. For example, CFHR1, most likely the main complement regulator in AMD pathogenesis was highly expressed in human RPE, but almost absent in mouse RPE. Furthermore, functions assigned to mouse and human RPE expression profiles indicate (patho-) biological differences related to AMD, such as oxidative stress, Bruch's membrane, immune-regulation and outer blood retina barrier.

Conclusion: These differences may be important for the development of new therapeutic strategies and translational studies in age-related macular degeneration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0141597PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627757PMC
June 2016