GAINING INSIGHT INTO THE PATHOLOGY OF 4H LEUKODYSTROPHY USING PATIENT-SPECIFIC IPSC-BASED MODELS Liza M. L. Kok
Cover design by Liza M. L Kok “Brain and bee hive side-by-side” Cover explanation: The brain is a complex organ, composed of many specialized cell types that work together to function harmoniously. To me, this has similarities with a beehive as bees in the hive have specialized jobs and the entire colony depends on each job being fulfilled correctly. One bee, the queen, has a clear function: to lay eggs. Nowadays, this is common knowledge, but for long it was thought that the big bee, is the ruler, and hence it must be the king! They couldn’t have been more wrong. Similarly, in this thesis, we sought to uncover which cell in the hypomyelinated 4H brain isn’t performing its role correctly. Rather than focusing solely on the most obvious suspect, we broadened our investigation— because, as with the bees, what seems logically is not always true! Layout: Liza M. L. Kok Printing: Ridderprint, www.ridderprint.nl ISBN: 978-94-6522-527-2 Copyright © 2025 L.M.L. Kok Publication of this thesis was financially supported by the Graduate School Neurosciences Amsterdam Rotterdam (ONWAR)
VRIJE UNIVERSITEIT GAINING INSIGHT INTO THE PATHOLOGY OF 4H LEUKODYSTROPHY USING PATIENT-SPECIFIC IPSC-BASED MODELS ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad Doctor of Philosophy aan de Vrije Universiteit Amsterdam, op gezag van de rector magnificus prof.dr. J.J.G. Geurts, volgens besluit van de decaan van de Faculteit der Bètawetenschappen in het openbaar te verdedigen op vrijdag 19 september 2025 om 9.45 uur in de universiteit door Liza Martha Linda Kok geboren te Wervershoof
promotoren: prof.dr. V.M. Heine prof.dr. N.I. Wolf promotiecommissie: prof.dr. R.E. van Kesteren dr. S. Dooves prof.dr. N. Nadif Kasri prof.dr. D. Pajkrt dr. N. Hamilton
TABLE OF CONTENTS Chapter 1 General introduction 7 Chapter 2 Cortical interneuron development is affected in 4H leukodystrophy Brain, 2023, 146(7), 2846-2860 35 Chapter 3 Investigating neuron intrinsic defects in 4H and Globoid Leukodystrophy 79 Chapter 4 Towards a 3D spheroid system for modelling leukodystrophies 109 Chapter 5 Human pluripotent stem cell-derived microglia shape neuronal morphology and enhance network activity in vitro Adapted from: Journal of Neuroscience Methods, 2025, Volume 415, 110354 155 Chapter 6 POLR3 gene and protein expression dynamics in 4H leukodystrophy using iPSC-derived neuronal lineages Under revision; Stem Cell Research, 2025 189 Chapter 7 Discussion 213 Appendix Summary Dankwoord / Acknowledgements 231 236
Chapter 1 8 The human brain is an incredibly complex organ, housing billions of interconnected neurons and glial cells that form highly specialized networks. Despite remarkable advancements in neuroscience, many of the brain’s functions and developmental processes remain only partially understood. What is clear, however, is that even minor deviations from normal brain development can lead to profound neurological and cognitive impairments. Among the many disorders that disrupt brain function are leukodystrophies—a diverse group of genetic diseases primarily affecting the brain’s white matter. One of the white matter components often affected is myelin, the insulating sheath that surrounds neuronal axons and facilitates efficient electrical signal transmission. One such leukodystrophy is 4H leukodystrophy, a disease that used to be characterized by hypomyelination along with hypodontia and hypogonadotropic hypogonadism. However, when in 2011 mutations in genes encoding for subunits of RNA polymerase III - a ubiquitous protein essential for cellular transcription - were identified as disease-causing variants, more patients with a wider range of clinical presentations were diagnosed. While the identification of the mutations provided an important first step toward understanding the genetic basis of 4H syndrome, it also raised critical questions about the underlying disease mechanisms and, most importantly, which cells should be the target of future treatments. Current treatments remain supportive, focusing on symptom management rather than addressing the root cause. Identifying which cell types are primarily affected and elucidating the processes disrupted by Pol III mutations are crucial steps toward developing targeted therapies. In this thesis, we aim to bridge this knowledge gap by modelling the cellular phenotypes of 4H leukodystrophy using human induced pluripotent stem cells (hiPSCs) and advanced in vitro systems. These models allow us to explore how Pol III defects affect brain cell development, particularly those involved in myelination and neuronal function. Through these studies, we seek to uncover disease mechanisms, identify vulnerable cell types, and evaluate potential therapeutic targets that could pave the way for future treatments.
Introduction 9 1 4H SYNDROME - Clinical spectrum Leukodystrophies are a large and heterogeneous group of genetic brain white matter disorders. Often they are diagnosed in childhood and have a progressive nature that leads to early death (van der Knaap et al., 2019). One of the more prevalent leukodystrophies is 4H syndrome (OMIM 612440) which is named after its characteristic clinical symptoms: hypomyelination, hypogonadotropic hypogonadism and hypodontia (Schmidt et al., 2020; Wolf, 2014). The first symptom, hypomyelination, is defined as a reduced amount or lack of normal myelin in the central nervous system (CNS), which can be diagnosed using MRI (Wolf et al., 2021). Patients often show cerebellar atrophy as well (Takanashi et al., 2014). Hypogonadotropic hypogonadism means that the hypothalamus or pituitary gland is not stimulating the testes or ovaries to produce sufficient sex hormones, leading to absent or abnormal puberty development (Fraietta et al., 2013). The last of the three typical symptoms is hypodontia, the absence of one or multiple primary or permanent teeth (Al-Ani et al., 2017). Even though these symptoms are characteristic for 4H, they are not always all present. The presentation of 4H ranges from very severe to mild and even asymptomatic young adults are described. The mildest patients present with isolated hypogonadotropic hypogonadism (Richards et al., 2017) while more severe patients seem to have a predominant neurological phenotype sometimes without typical hypomyelination (DeGasperis et al., 2020; Harting et al., 2020; La Piana et al., 2016; Wolf, 2014). There is not only heterogeneity in the presentation of the characteristic symptoms, there is also a wide spectrum of other symptoms and signs of the disease amongst which are other neurological presentations such as delay in motor development in early life and/or regression of motor development later in life, often resulting in wheelchair dependence. Additionally, many patients show cerebellar features such as ataxia and dysarthria. Later, pyramidal features such as the development of spasticity can be seen but this is more rare. Also non-neurological symptoms such as short stature, caused by endocrine abnormalities, and myopia (shortsightedness) are observed. Furthermore additional dental abnormalities like natal teeth, delayed dentition, and unusual order of tooth eruption are not uncommon. (Pelletier et al., 2021; Wolf, 2014). Craniofacial features may also be seen (Mirchi et al., 2023). A graphical representation of the wide spectrum of clinical symptoms is depicted in Figure 1.
Chapter 1 10 Figure 1: Clinical presentation of 4H syndrome. Neurological (blue) and non-neurological (white) clinical presentations. Created using BioRender.com. 4H SYNDROME – Current Treatments Due to its multisystem involvement, 4H leukodystrophy requires multidisciplinary care involving several subspecialists, including endocrinologists, neurologists, dentists, and clinical geneticists. Current treatment strategies are symptom-based, focusing on managing the broad range of clinical features associated with the disorder. Management of the three hallmark symptoms involves supportive care such as physical and occupational therapy for motor and neurological issues that might be caused by hypomyelination, hormone replacement therapy (HRT) for hypogonadotropic hypogonadism (Billington et al., 2015; Nwatamole et al., 2024), and specialized dental evaluations to address hypodontia. While these interventions can improve the quality of life for affected individuals, there is currently no curative therapy for 4H syndrome. Its progressive nature and clinical variability stress the urgent need for novel therapeutic strategies targeting and curing the underlying pathology. Identifying specific therapeutic targets at the molecular and cellular level will be crucial in developing future disease-modifying therapies. Advances in understanding 4H syndrome’s genetic and cellular basis hold promise for translating research findings into meaningful clinical interventions. In summary, 4H syndrome is a relatively prevalent leukodystrophy with a highly variable clinical presentation and progressive disease course. Current treatments remain supportive, focusing on symptom management and improving patients’ quality of life, but they lack curative potential (Adang et al., 2017). To provide novel perspectives new curative treatment strategies are urgently needed. To develop these, we need to understand the targets for therapies.
Introduction 11 1 4H SYNDROME - Genetic basis The identification of the genetic basis of 4H leukodystrophy marked a critical step toward understanding the disease and developing targeted therapies. Prior to 2011, patients were diagnosed solely based on clinical features, with no molecular explanation for the disease (Timmons et al., 2006; Wolf et al., 2005; Wolf et al., 2007). This changed with the discovery of mutations in genes encoding subunits of RNA polymerase III (Pol III), a key enzyme involved in the transcription of small, non-coding RNAs essential for cellular function. The first identified mutations were identified in the POLR3A gene, which encodes one of the 17 subunits of Pol III, followed shortly by the identification of mutations in POLR3B, which encodes another subunit of the enzyme's catalytic core (Bernard et al., 2011; Tetreault et al., 2011). These findings established Pol III as the central molecular player in the disease. In subsequent studies, rarer mutations were discovered in additional Pol III subunits, including POLR3K and POLR3D, as well as POLR1C, a shared subunit between Pol III and RNA polymerase I (Dorboz et al., 2018; Macintosh, Perrier, et al., 2023; Thiffault et al., 2015). Since all the identified mutations for 4H syndrome are related to genes that encode for subunits of Pol III (Figure 2) (Gauquelin et al., 2019; Wolf, 2014), the disease is also referred to as POLR3-related leukodystrophy. The pathogenic variants observed in these genes are diverse, both in genomic location and mutation type. They include missense, nonsense, and splice-site mutations, as well as small intragenic deletions, deep intronic variants, insertions, and large multiexon deletions (Bernard et al., 2011; Daoud et al., 2013; Gutierrez et al., 2015; Hiraide et al., 2020; La Piana et al., 2016; Potic et al., 2012; Saitsu et al., 2011; Tetreault et al., 2011; Wolf, 2014). The wide range of mutation types highlights the molecular complexity of the disease and suggests the possible need for patient- or mutation-specific therapeutic approaches. Figure 2: Schematic representation of RNA Polymerase III an I. Subunits in blue are related to 4H syndrome. Figure adapted from (Thiffault et al., 2015)
Chapter 1 12 RNA POL III - In health To comprehend the consequences of mutations in RNA polymerase III (Pol III) subunits, it is essential to understand its function under physiological conditions. Pol III is one of three eukaryotic RNA polymerases that transcribe DNA into RNA, each with distinct transcriptional specializations. Pol I transcribes pre-rRNAs, which are processed into the 18S, 5.8S, and 28S ribosomal RNAs (rRNAs), essential components of ribosome assembly. Pol II transcribes protein-coding genes into mRNAs and produces several regulatory RNAs, including most microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). In contrast, Pol III transcribes small, highly abundant non-coding RNAs (ncRNAs), typically less than 350 nucleotides in length, that are essential for various cellular processes. Among Pol III-transcribed transcripts are nuclear transfer RNAs (tRNAs) and 5S rRNA, which are both integral components of translation (Hoagland et al., 1958; Rosset & Monier, 1963). Ribosome binding and translation regulation are also mediated by Pol III transcripts, specifically, 7SL, BC200, snaR and Alu (Bishop et al., 1970; Houck et al., 1979; Mrazek et al., 2007; Parrott & Mathews, 2007; Watson & Sutcliffe, 1987). Other essential Pol III products are 7SK RNA, U6 and U6atac which are involved in transcription regulation and splicing (Lerner & Steitz, 1979; Zieve & Penman, 1976). Furthermore, transcripts RPPH1 and RMRP are key to tRNA and ribosomal RNA processing, while Y RNA plays a role in RNA stability, replication and translation regulation (Bartkiewicz et al., 1989; Lerner et al., 1981; Reddy et al., 1981). Also vault ribonucleoprotein (RNP) regulation of autophagy is mediated by Pol III transcript, vault RNA (Kedersha & Rome, 1986). Finally, Pol III transcript nc886 is involved in immune signalling (Mrazek et al., 2007; Nandy et al., 2009; Zhou & Van Bortle, 2023). Figure 3: Overview of RNA polymerase III transcriptome. Reused from (Zhou & Van Bortle, 2023)
Introduction 13 1 These examples illustrate that the Pol III transcriptome is extensive and supports a diverse range of fundamental cellular processes. However, it remains unclear if and how these transcripts and the processes they are involved in are affected by 4H related POLR3 variants. RNA POL III - In 4H leukodystrophy The identification of the causal genes for 4H syndrome significantly improved diagnostic capabilities, yet it raised the question of how mutations in Pol III—a protein that is abundantly expressed and essential for transcription—result in this disease. Despite ongoing research, the precise mechanisms underlying the pathogenesis of 4H leukodystrophy remain unclear. One proposed mechanism is that certain mutations disrupt the structure, complex formation, or cellular localization of Pol III. For example, mutations in POLR1C have been shown to impair the assembly and nuclear import of RNA Pol III (Thiffault et al., 2015). Similarly, mutations in POLR3B that map to conserved protein domains have been hypothesized to interfere with DNA binding, modify the catalytic cleft, or disrupt subunit interactions (Tetreault et al., 2011). Experimental studies have shown that a specific POLR3B variant affected complex assembly, that could be restored by riluzole administration (Pinard et al., 2022). Another POLR3B variant caused aberrant association of individual enzyme subunits rather than affecting overall enzyme assembly (Choquet, Pinard, et al., 2019; Djordjevic et al., 2021). Another potential pathological mechanism in 4H involves alterations in POLR3 gene and/or protein expression. In various samples of patients with POLR3A variants, amongst which fibroblasts, grey and white matter tissue, decreased Pol IIIA subunit expression has been reported (Bernard et al., 2011; Perrier, Gauquelin, et al., 2020). Similarly, a decrease in POLR3 gene expression has been described in patients with POLR3D variants (Macintosh, Perrier, et al., 2023). Additionally, mutant clones harbouring 4H-related POLR3A variants showed reduced Pol IIIA protein and POLR3A mRNA levels, however this was not consistent across all clones (Choquet, Forget, et al., 2019). For POLR3B variants, decreased gene levels have been described as well (Mattijssen et al., 2024). The afore mentioned findings have only been reported in small cohorts, often linked to variants causing severe phenotypes. This leaves uncertainty about whether these findings are universal.
Chapter 1 14 In line with molecular disruptions of the affected subunits, several studies have documented reduced Pol III transcript levels in various patient-derived samples. Studies have shown that Pol III products such as tRNAs and BC200, which are involved in translation, are dysregulated in patient-derived fibroblasts and cellular models of 4H (Choquet, Forget, et al., 2019). Similar reductions have been observed in fibroblasts from patients with POLR3K variants, where tRNAs, H1 RNA, 5S rRNA, and 7SL RNA were significantly downregulated (Dorboz et al., 2018). Reduced levels of POLR3 transcripts have also been detected in blood RNA samples from patients with POLR3A variants (Azmanov et al., 2016). A mouse model with POLR3A variants further supports this, showing reduced tRNA levels (Moir et al., 2024). It is also shown in yeast that mutations associated with 4H decrease tRNA transcription (Arimbasseri et al., 2015). Tissue-specific vulnerability to Pol III mutations may also contribute to 4H syndrome’s pathology. For instance, it is hypothesized that oligodendrocytes, which require substantial protein synthesis during myelination, might be particularly affected due to the role of Pol III transcripts in translation (Perrier, Michell-Robinson, et al., 2020). Considering that Pol III products like tRNAs, have tissue specific expression profiles makes this hypothesis even more plausible (Dittmar et al., 2006). Additionally, the introduction of a POLR3A variant in MO3.13 cells (human oligodendrocytes) reduced myelin basic protein (MBP) levels in these oligodendrocytes (Choquet, Forget, et al., 2019), further pointing towards oligodendrocyte vulnerability. Additional evidence is found in the fact that OPCs with decreased RNA Pol III subunit expression showed altered oligodendrocyte differentiation, maturation and myelination capacity (Macintosh, Michell-Robinson, et al., 2023). Similarly, a mouse model with POLR3B exon 10 deletion exhibited defects in oligodendrocyte proliferation and differentiation, supporting the role of Pol III in myelination (Michell-Robinson et al., 2023). However, these mechanisms focus mainly on oligodendrocytes, this does not provide an explanation for 4H patients presenting mainly with neurological symptoms. An alternative hypothesis involves cell-type-specific effects mediated by alternative splicing. For instance, the intronic variant c.1771-7C>G in POLR3A leads to altered transcripts, including the skipping of exon 14, resulting in a premature stop codon or an in-frame deletion of exons 13 and 14 (Perrier, Gauquelin, et al., 2020) Similarly, POLR3A variant c.1771-6C>G is reported to result in exon 14 skip (Azmanov et al., 2016; Yoon Han et al., 2022). And also for POLR3B variants such as c.1625A>G and c.2084-6A>G, mis-splicing has been reported (Daoud et al.,
Introduction 15 1 2013; Mattijssen et al., 2024). Another POLR3-related disorder, caused by POLR3GL variants, also has splicing alteration (Terhal et al., 2020). While significant progress has been made in characterizing the molecular and cellular effects of Pol III mutations, and many hypotheses on the disease mechanisms have been formed, no treatment targets have been yet identified. Understanding why certain tissues and cell types are more severely affected in 4H will be critical to develop targeted therapeutic strategies. CURRENT 4H DISEASE MODELS To advance knowledge on 4H leukodystrophy, it is essential to develop robust disease models that can capture its complex cellular and molecular phenotypes, enabling the investigation of tissue-specific vulnerability and potential therapeutic targets. The field has made several efforts in this direction. Early attempts using mouse models with known 4Hassociated POLR3A variants such as c.2015G>A (p.Gly672Glu), did however not have neurological abnormalities or Pol III transcript alterations. Additionally, Polr3a−/−null mice were embryonic lethal (Choquet et al., 2017). Similarly, a mouse model for POLR3B variant c.308G>A was embryonic lethal when homozygous. While a double-mutant (Polr3aG672E/null Polr3b+/R103) did not show neurological or transcription abnormalities (Choquet, Pinard, et al., 2019). Recent advancements have overcome these problems, and now mouse models harbouring Polr3a and Polr3b variants are available and reflect leukodystrophy phenotypes (Merheb et al., 2021; Michell-Robinson et al., 2023; Moir et al., 2024) These new disease models are invaluable tools to progress research on 4H leukodystrophy but the absence of phenotypes in the earlier mouse models shows that vulnerability to Pol III mutations likely varies between species, underscoring the need for human-derived models. Considering the many variants causing 4H and the broad clinical spectrum, a patient- and/or mutation-specific approach may be necessary to fully capture the complexity of the disease. Currently, the availability of human tissue samples from individuals with 4H leukodystrophy is limited. Post-mortem tissue is scarce and typically obtained only at the end stage of the disease, providing limited insight into early disease processes and progression—key aspects for therapy development. While patient-derived fibroblasts have been used, these are not the cells that show defects in 4H. MO3.13 oligodendroglial cell models have been used in
Chapter 1 16 some studies, but do not fully recapitulate the complex neurodevelopmental environment of the human brain. Therefore, more physiologically relevant models that mimic early tissue development are needed to investigate cell-specific vulnerabilities in 4H and identify early potential therapeutic targets. HUMAN INDUCED PLURIPOTENT STEM CELLS To overcome these limitations, human pluripotent stem cells have emerged as a powerful alternative. Pluripotent stem cells have the unique ability to self-renew and differentiate into cell types from all three germ layers: endoderm, ectoderm and mesoderm. In vivo, pluripotent cells are found in embryos (Thomson et al., 1998). However, by inducing the overexpression of specific transcription factors, somatic cells can be reprogrammed into a pluripotent state, creating human induced pluripotent stem cells (hiPSCs) (Takahashi et al., 2007). These hiPSCs retain the genetic profile of the donor cells and can be differentiated into virtually any cell type, making them particularly suitable for investigating the molecular and cellular mechanisms underlying 4H leukodystrophy. The versatility of pluripotent stem cells lies in their capacity to generate virtually any cell type through directed differentiation. But now the question emerges, which tissue will be investigated? KEY PLAYERS IN HYPOMYELINATION AND NEUROLOGICAL DEFICITS IN 4H Identifying relevant tissues and cell types is essential for understanding the pathophysiology of 4H leukodystrophy and developing targeted therapies. Since 4H usually is characterized primarily by hypomyelination and associated neurological deficits, our focus is on modelling early brain development—the stage when many patients begin to show their first symptoms. This stage of development aligns with essential neurodevelopmental processes such as gliogenesis, myelination and refinement of synapses and circuits (Zhou et al., 2024). The challenge, however, lies in replicating these highly dynamic processes of human brain development in vitro. To gain meaningful insights into 4H pathophysiology, cellular models must reflect aspects of these complex interactions while enabling the study of individual cell-type vulnerabilities. Hence, we focused on a variety of models that centre around key
Introduction 17 1 brain cell types which are known to be involved in different genetic white matter disorders. The following sections provides a brief introduction to the cell lineages we aimed to investigate, and their known involvements in genetic white matter disorders. Oligodendrocytes The primary cells responsible for myelination are oligodendrocytes, which originate from NG2-expressing cells known as polydendrocytes or oligodendrocyte precursor cells (OPCs). Mature oligodendrocytes extend multiple protrusions that wrap around axons, forming a lipid-dense myelin sheath. This sheath enhances electrical signal conduction while also providing metabolic support, regulating ion and water homeostasis, and adapting to activity-dependent neuronal signals (Kuhn et al., 2019). Within genetic white matter disorders, some have primary myelin pathology, and hence are classified as myelin disorders (van der Knaap & Bugiani, 2017). Within these myelin disorders, three subcategories can be distinguished based on the type of myelin pathology: hypomyelination, demyelination and myelin vacuolization. An example of a hypomyelinating white matter disorder is Pelizaeus-Merzbachter disease, which is caused by variants in PLP1, a gene that is mainly expressed in mature oligodendrocytes (Torii et al., 2014). Metachromatic leukodystrophy (MLD) and Globoid cell leukodystrophy (GLD) also known as Krabbe disease and the cerebral form of X-linked Adrenoleukodystrophy are characterized by demyelination. Those disease are respectively caused by variants in ASA, GALC and ABCD1 and cause pathological accumulation of respectively sulfatides and psychosine due to lysosomal storage disorders and very long chain fatty acids due to peroxisomal dysfunction (Abed Rabbo et al., 2021; Berger et al., 2014). The last category is characterized by myelin vacuolization and among this falls the leukodystrophy Canavan disease. Canavan disease is caused by ASPA variants a gene that codes for the substrate enzyme aspartoacylase, which hydrolyzes N-acetylaspartic acid (NAA) to acetate and aspartate (Hoshino & Kubota, 2014). Neurons Since oligodendrocytes myelinate neuronal axons, neurons are crucial in the mimicking of (hypo)myelination. Neurons transmit information through voltage discharges along their cell membranes. Depending on the neurotransmitters they release at the synapse, neurons are broadly divided into excitatory/glutamatergic or inhibitory/GABAergic subtypes. Certain neuronal subtypes, such as inhibitory parvalbumin-expressing neurons, are particularly likely to be myelinated (Stedehouder et al., 2017). A subclass of genetic white matter
Chapter 1 18 disorders, the leuko-axonopathies, are due to defects in neuron- or axon-specific gene products or they have central disease mechanisms which can be conducted back to axons (van der Knaap & Bugiani, 2017). For example, hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is caused by variants in the TUBB4A gene, a gene coding for microtubules highly expressed in the brain (Hamilton et al., 2014). For others such as 4H leukodystrophy the affected gene is generic, but histopathology shows widespread axonal damage, pointing towards important neuroaxonal involvement. However, many end stage brain disorders involve neuronal damage without providing insight in the initial problems (Wolf, 2014). Astrocytes While neurons and oligodendrocytes have obvious roles in myelination, other brain cell types, such as astrocytes, also play crucial roles. Astrocytes perform various essential functions, including maintaining brain homeostasis, supporting the blood-brain barrier, and regulating neurotransmitter and lipid metabolism (Hasel et al., 2023). They are morphologically and functionally diverse, exhibiting regional differences in identity and specialization. Some genetic white matter disorders are known to be caused by variants in astrocyte-specific genes or in which astrocytes play crucial roles in the disease mechanisms and are referred to astrocytopathies (van der Knaap & Bugiani, 2017). For example, Alexander disease is a rare genetic astrocytopathy caused by variants in GFAP, which codes for glial fibrillary acidic protein (GFAP), an astrocyte-specific cytoskeletal intermediate filament protein (Brenner et al., 2001). For vanishing white matter (VWM) the causative gene is not astrocyte-specific but astrocytes are still central in the pathomechanisms of vanishing white matter. In vitro cultures have shown that VWM astrocytes secreted factors affect the oligodendrocyte lineage (Dooves et al., 2016). Microglia Microglia may also have an indirect role in 4H-related hypomyelination. Unlike other brain cells, microglia are thought to originate from erythro-myeloid progenitors (EMPs) in the developing yolk sac, making them of mesodermal lineage. Microglia play a dual role in immune defence and brain homeostasis by regulating neurogenesis, promoting neuronal survival, and performing synaptic pruning, thereby enhancing neural network efficiency and maturation (Paolicelli & Ferretti, 2017). Emerging research has begun to explore microglial in 4H leukodystrophy (Moir et al., 2024). Microgliopathies are observed in some white matter disorders, specifically CSF1R-related disorders, of which the gene is mainly expressed in
Introduction 19 1 microglia, and Nasu-Hakola disease (Sasaki, 2017). But allogeneic hematopoetic stem cell transplantation (HSCT), thought to replace microglia, has been shown successful in other white matter disorders, specifically in the cerebral from of X-linked adrenoleukodystrophy, metachromatic leukodystrophy and Krabbe disease. This shows the potential involvement of microglia in more leukodystrophies and the potential of microglia as therapeutic strategy (Page et al., 2019). The human brain comprises highly specialized cell types with diverse functions and complex interactions. Although oligodendrocytes are likely central to 4H-associated hypomyelination, it is essential to consider the broader neural network, including neurons, astrocytes, and microglia as these have proven to be deficient in other genetic white matter disorders. Understanding how these cell types interact in 4H could provide valuable insight into the mechanisms underlying 4H leukodystrophy and guide the development of therapeutic strategies. IPSC-DERIVED MODELS FOR 4H RESEARCH Given the limited availability of patient-derived brain tissue, human-induced pluripotent stem cells (hiPSCs) provide a valuable platform for modelling 4H leukodystrophy. Since 4H pathology originates during early brain development, our in vitro models focus on recapitulating key developmental stages through directed differentiation protocols. These include generating relevant brain cell types using classical patterning protocols, creating co-cultures, and establishing three-dimensional (3D) brain organoids. Classical Patterning for Directed Differentiation Classical patterning protocols aim to recapitulate embryonic development by exposing pluripotent stem cells to specific molecular cues that guide them toward neural fates. This method allows the generation of cell types at sequential developmental stages, reflecting the physiological processes occurring during early brain development. For example, dual SMAD inhibition using dorsomorphin and SB431542 is commonly used to promote neural lineage commitment, inducing a neuroectodermal fate that serves as a starting point for neuronal and glial cell lineages (Nadadhur et al., 2017; Shi et al., 2012). While classical patterning is slow compared to overexpression protocols it preserves the natural progression of cell maturation, making it a valuable tool for studying disorders that manifest during specific developmental stages, such as 4H (Zhang et al., 2013) (Yang et al., 2017).
Chapter 1 20 Combining Cell Types: Towards More Physiological Models To better reflect the complex, interconnected nature of the human brain, differentiated cell types can be combined into co-culture systems. This approach allows for the study of cellcell interactions relevant to myelination and neuronal network formation. For instance, neurons and glia can be differentiated separately and later co-cultured at specific maturation stages, enabling the investigation of glial support or impairment to neuron development in 4H (Dooves et al., 2019). This strategy provides an opportunity to model disease-specific cellular interactions while maintaining experimental flexibility. Alternatively, a more integrated approach involves generating three-dimensional (3D) brain-like structures known as organoids. These self-organizing cultures emerge from pluripotent stem cells when exposed to patterning cues that guide them toward specific brain regions (Lancaster et al., 2013). Organoids offer a more comprehensive representation of brain development, enabling multi-lineage differentiation and spatial organization. Offering a unique platform for studying complex, multi-cellular interactions in 4H leukodystrophy. Given the importance of replicating early brain development in studying 4H leukodystrophy, this thesis employs a combination of classical patterning-based monocultures, co-cultures, and brain organoids. By leveraging these models, we aim to uncover how key brain cell types contribute to the disease's pathophysiology, including hypomyelination and related neurological deficits. This multi-faceted approach provides a platform not only to investigate the molecular and cellular basis but hopefully also helps to pinpoint potential therapeutic targets. Insights gained from these models could inform future treatment strategies, including gene and cell replacement therapies, and the models could be used for high-throughput drug screening for disease-modifying compounds. BRIDGING RESEARCH TO TREATMENT DEVELOPMENT While restorative treatments have been developed for some leukodystrophies (Krivit, 2004; Krivit et al., 1999; van den Broek et al., 2018), no such therapies currently exist for 4H. Possibly gene replacement or correction of endogenous mutations could restore Pol III function. Similarly, cell replacement therapy, involving donor cells or ex vivo gene-corrected cells, could replenish damaged cell populations. However, developing these therapies requires a deep understanding of which cell types are primarily affected and need to be replaced or corrected.
Introduction 21 1 Less invasive treatment options such as drug therapies that target disrupted pathways could potentially slow or halt disease progression. Identifying such pathways and screening for compounds that alter the pathways requires realistic in vitro models that accurately replicate 4H pathology—models that are currently lacking. By establishing such models, this thesis aims to close this gap, facilitating both investigating of therapeutic targets as well as development of models that might be used in future therapeutic screening. This does not only apply to 4H, but might also be used for other leukodystrophies. THESIS OUTLINE In this thesis we aim to gain insight into how molecular and cellular mechanisms contribute to the pathology of 4H leukodystrophy using patient-specific iPSC-based models with the goal to facilitate development of new therapeutic strategies. To address this question and also to unravel patient-specific genetics contributing to broad clinical presentations, we used iPSC lines from patients with either POLR3A or POLR3B variants, that are exonic or intronic and which are relatively common in the 4H patient population (Figure 4). To investigate if our findings are unique to 4H leukodystrophy or could be translated to other leukodystrophies, some studies also include iPSCs of other leukodsytrophies. Here we provide the research questions, approaches and main results of the following experimental chapters: Chapter 2: First we explored what genes are dysregulated in brain specific cell types in 4H patient cultures using patient iPSC technology in combination with RNAseq analysis on iPSC-derived cerebellar cells. Downregulation of ARX, a gene involved in early brain and interneuron development gave first suggestions to further explore interneuron dysfunction in 4H leukodystrophy. In cortical neuron cultures containing both glutamatergic and GABAergic neuron populations, we confirmed the downregulation of ARX in 4H and a reduction in GABAergic synapses. Functional analysis by MEA further pointed towards alterations in the inhibitory neuron population. Initial analysis points to no obvious intrinsic changes in the oligodendrocyte population. Since ARX is an important regulator of SHH gradients, we investigated whether targeting the SHH pathway could improve 4H associated GABAergic phenotypes, but no improvements were observed in our assays. In conclusion, we identified cortical interneuron development to be affected in 4H leukodystrophy.
Chapter 1 22 Figure 4: Overview of 4H mutations within this cohort and their evolutionary conservation. Schematic representation of mutations in 4H patient cohort in A) POLR3A gene and B) POLRB gene. Open triangles intron mutations, filled triangles exon mutations. Overview of genetic conservation for C) POLR3A and D) POLR3B intron variants. E) Reconstruction of protein Pol III A and B subunit with DNA (black) and RNA (gray), and exon mutations (red circles) according to PDB ID: 7DN3 Human RNA Polymerase III elongation complex, images created using Mol* (Li et al., 2021; Sehnal et al., 2021). Conservation of amino acid sequences per mutation in F) Pol III A and G) B subunit. Conservation according to MultiZ alignment of the UCSC Genome Browser on Human GRCh38/hg38 (Nassar et al. 2023). Species: Homo sapiens (human), Mus musculus (mouse), Danio rerio (zebrafish), Drosophila melanogaster (fruit fly), Caenorhabditis elegans (roundworm) and Saccharomyces cerevisiae (yeast). “=” means aligning species has one or more unalignable bases in the gap region, “-“ no bases in the aligned species. Chapter 3: To further explore neuronal phenotypes in 4H leukodystrophy and whether changes are disease-specific or more generally affected in leukodystrophies, we studied key phenotypic and transcriptomic differences between Ctrl, 4H and GLD neurons. By comparing neuronal changes in 4H and GLD, this chapter highlights the heterogeneity of leukodystrophies and the necessity of refining disease models. The findings underscore a
Introduction 23 1 role for neuronal dysfunction in 4H, expanding the focus beyond traditional glial-centric paradigms. Chapter 4: To study changes in neuron-glia interactions in 4H leukodystrophy and leukodystrophies in general, we developed an EU platform based on myelinating spheroids. As no existing standardized platform to investigate leukodystrophies was in place, we distributed iPSCs for the centralized generation of myelinating spheroids of GLD, CD, 4H and controls. We explored whether these 3D spheroids generate the cellular composition required for the investigation of leukodystrophies, including neurons, oligodendrocytes, and astrocytes. We confirmed relevance for investigating leukodystrophies and report differential expressed genes and significant gene sets that can guide future research directions. In conclusion, this chapter validated the potential of 3D spheroid models for studying leukodystrophies by mimicking disease-relevant cellular interactions. It emphasizes their utility in identifying cell-type-specific vulnerabilities and novel molecular targets, addressing the gap in comprehensive in vitro models. Chapter 5: In the myelinating spheroids, one cell type was not included, the microglia. Hence, we generated a platform for the co-culture of iPSC-derived microglia-like cells and analysed the effect of the microglia on the neuronal cultures using cellomics and multielectrode arrays (MEA). The addition of microglia significantly impacted the cultures. ALD microglia seemed to affect axons different compared to control microglia, while 4H microglia seemed to function fine. Chapter 6: To explore how patient-specific genetic variants affect POLR3 gene and protein expression during neuronal lineage differentiation in 4H leukodystrophy, we examined POLR3 expression levels, protein localization, and developmental dynamics across iPSCs, neural epithelial cells and neurons. We also evaluated whether the genetic background contributes to expression differences. This chapter integrates patient-specific data with iPSC models to elucidate disease mechanisms. By addressing genetic variability, this approach paves the way for precision medicine approaches in 4H leukodystrophy research. Chapter 7: In this concluding chapter, we evaluate the model systems used throughout the thesis, discussing their advantages, limitations, and suitability for future leukodystrophy research. We provide an overarching synthesis of the knowledge gained from each chapter and how these findings interconnect. Additionally, we present forward-looking perspectives
Chapter 1 24 on how these models could advance toward clinical trials. We explore whether the models and findings generated in this thesis could support the development of targeted therapies and inform future clinical applications.
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