Hao-Fountain Syndrome (HAFOUS) is an autosomal dominant neurodevelopmental disorder caused by pathogenic variants in the USP7 gene. Research into the molecular mechanisms of HAFOUS has been limited by the lack of suitable human model systems. In this study, we introduce induced pluripotent stem cell (iPSC) lines derived from a HAFOUS patient, providing a valuable model to investigate mechanisms of altered cell lineage commitment during disease progression. This model offers a platform to explore the role of USP7 variants in HAFOUS pathogenesis and develop potential therapeutic strategies.

Clark-Baraitser Syndrome (CLABARS) is an autosomal dominant neurodevelopmental disorder caused by pathogenic variants in the TRIP12 gene. Research into the molecular mechanisms of CLABARS has been limited by the lack of suitable human model systems. In this study, we introduce an induced pluripotent stem cell (iPSC) line derived from a CLABARS patient, providing a promising model to investigate mechanisms of altered cell lineage commitment during disease progression.

Autism spectrum disorder (ASD) is characterized by difficulties in social interaction, communication, and repetitive behavior, typically diagnosed during early childhood and attributed to altered neuronal network connectivity. Several genetic and environmental risk factors contribute to ASD, including pre- or early life immune activation, which can trigger microglial and astroglial reactivity, impacting early neurodevelopment. In ASD, astrocytes show altered glutamate metabolism, directly influencing neuronal network activity, while microglia display impaired synaptic pruning, an essential developmental process for the refinement of neuronal connections. Additionally, reduced myelination in specific cortical and subcortical regions may affect brain connectivity in ASD, with white matter integrity correlating with the severity of the disorder, suggesting an important role for oligodendrocytes and myelin in ASD. This chapter provides an overview of current literature on the role of neuroglia cells in ASD, with a focus on immune activation, glutamate signaling, synaptic pruning, and myelination.

4H leukodystrophy is a rare genetic disorder classically characterized by hypomyelination, hypodontia and hypogonadotropic hypogonadism. With the discovery that 4H is caused by mutations that affect RNA polymerase III, mainly involved in the transcription of small non-coding RNAs, patients with atypical presentations with mainly a neuronal phenotype were also identified. Pathomechanisms of 4H brain abnormalities are still unknown and research is hampered by a lack of preclinical models. We aimed to identify cells and pathways that are affected by 4H mutations using induced pluripotent stem cell models. RNA sequencing analysis on induced pluripotent stem cell-derived cerebellar cells revealed several differentially expressed genes between 4H patients and control samples, including reduced ARX expression. As ARX is involved in early brain and interneuron development, we studied and confirmed interneuron changes in primary tissue of 4H patients. Subsequently, we studied interneuron changes in more depth and analysed induced pluripotent stem cell-derived cortical neuron cultures for changes in neuronal morphology, synaptic balance, network activity and myelination. We showed a decreased percentage of GABAergic synapses in 4H, which correlated to increased neuronal network activity. Treatment of cultures with GABA antagonists led to a significant increase in neuronal network activity in control cells but not in 4H cells, also pointing to lack of inhibitory activity in 4H. Myelination and oligodendrocyte maturation in cultures with 4H neurons was normal, and treatment with sonic hedgehog agonist SAG did not improve 4H related neuronal phenotypes. Quantitative PCR analysis revealed increased expression of parvalbumin interneuron marker ERBB4, suggesting that the development rather than generation of interneurons may be affected in 4H. Together, these results indicate that interneurons are involved, possibly parvalbumin interneurons, in disease mechanisms of 4H leukodystrophy.

Introduction Vanishing white matter (VWM) is a leukodystrophy that leads to neurological dysfunction and early death. Astrocytes are indicated as therapeutic target, because of their central role in VWM pathology. Previous cell replacement therapy using primary mouse glial precursors phenotypically improved VWM mice. Aims The aim of this study was to determine the translational potential of human stem cell-derived glial cell replacement therapy for VWM. We generated various glial cell types from human pluripotent stem cells in order to identify a human cell population that successfully ameliorates disease hallmarks of a VWM mouse model. The effects of cell grafts on motor skills and VWM brain pathology were assessed. Results Transplantation of human glial precursor populations improved the VWM phenotype. The intrinsic properties of these cells were partially reflected by cell fate post-transplantation, but were also affected by the host microenvironment. Strikingly, the spread of transplanted cells into the white matter versus the gray matter was different when grafted into the VWM brain as compared to a healthy brain. Conclusions Transplantation of human glial cell populations can have therapeutic effects for VWM. For further translation to the clinic, the microenvironment in the VWM patient brain should be considered as an important moderator of cell replacement therapy.

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.

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, Dictyostelium, 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 4Ebinding 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 Dictyostelium 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 highglucose 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.

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.

Astrocytes gained attention as important players in neurological disease, including a number of leukodystrophies. Several studies explored the generation of induced pluripotent stem cell-derived astrocytes for drug screening and regenerative studies. Developing robust models of patient induced pluripotent stem cells is challenged by high variability due to diverse genetic backgrounds and long-term culture procedures. While human models are of special interest, mouse-based models have the advantage that for them these issues are less pronounced. Here we present astrocyte differentiation protocols for both human and mouse induced pluripotent stem cells to specifically induce grey and white matter astrocytes. Both subtypes expressed astrocyte-associated markers, had typical astrocyte morphologies, and gave a reactive response to stress. Importantly, the grey and white matter-like astrocytes differed in size, complexity of processes, and expression profile, conform primary grey and white matter astrocytes. The newly presented mouse and human stem cell-based models for the leukodystrophy Vanishing White Matter replicated earlier findings, such as increased proliferation, decreased OPC maturation and modulation by hyaluronidase. We studied intrinsic astrocyte subtype vulnerability in Vanishing White Matter in both human and mouse cells. Oligodendrocyte maturation was specifically inhibited in cultures with Vanishing White Matter white matter-like astrocytes. By performing RNA sequencing, we found more differentially regulated genes in the white than in the grey matter-like astrocytes. Human and mouse astrocytes showed the same affected pathways, although human white matter-like astrocytes presented human-specific disease mechanisms involved in Vanishing White Matter. Using both human and mouse induced pluripotent stem cells, our study presents protocols to generate white and grey matter-like astrocytes, and shows astrocyte subtype-specific defects in Vanishing White Matter. While mouse induced pluripotent stem cell-based cultures may be less suitable to mimic human astrocyte subtype- or patient-specific changes, they might more robustly represent disease mutation-related cellular phenotypes as the cells are derived from inbred mice and the protocols are faster. The presented models give new tools to generate astrocyte subtypes for in vitro disease modeling and in vivo regenerative applications.

ObjectiveAstrocytes 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.MethodsHere, 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.ResultsWe 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.InterpretationAstrocyte 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

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 cocultured 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.

Aim: Vanishing White Matter (VWM) is a devastating leucoencephalopathy without effective treatment options. Patients have mutations in the EIF2B1-5 genes, encoding the five subunits of eIF2B, a guanine exchange factor that is an important regulator of protein translation. We recently developed mouse models for VWM that replicate the human disease. To study disease improvement after treatment in these mice, it is essential to have sensitive biomarkers related to disease stage. The Bergmann glia of the cerebellum, an astrocytic subpopulation, translocate into the molecular layer in symptomatic VWM mice and patients. This study looked at the prospects of using Bergmann glia pathology as an objective disease marker for VWM. Methods: We defined a new quantitative measurement of Bergmann glia pathology in the cerebellum of VWM mice and patients. To test the sensitivity of this new marker for improvement, VWM mutant mice received long-term treatment with Guanabenz, an FDAapproved anti-hypertensive agent affecting eIF2B activity. Results: Bergmann glia translocation was significantly higher in symptomatic VWM mice and VWM patients than in controls and worsened over the disease course. Both Bergmann glia pathology and cerebellar myelin pathology improved with Guanabenz treatment in mice, showing that Bergmann glia translocation is a sensitive measurement for improvement. Conclusions: Bergmann glia translocation can be used to objectively assess effects of treatment in VWM mice. Future treatment strategies involving compounds regulating eIF2 phosphorylation might benefit VWM patients.

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.

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.

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.

The proteasome is the major protein degradation system within the cell, comprised of different proteolytic subunits; amyloid-beta is thought to impair its activity in Alzheimer's disease. Neuroinflammation is a prominent hallmark of Alzheimer's disease, which may implicate an activation of the immunoproteasome, a specific proteasome variant induced by immune signalling that holds slightly different proteolytic properties than the constitutive proteasome. Using a novel cell-permeable proteasome activity probe, we found that amyloid-beta enhances proteasome activity in glial and neuronal cultures. Additionally, using a subunit-specific proteasome activity assay we showed that in the cortex of the APPswePS1dE9 plaque pathology mouse model, immunoproteasome activities were strongly increased together with increased messenger RNA and protein expression in reactive glia surrounding plaques. Importantly, this elevated activity was confirmed in human post-mortem tissue from donors with Alzheimer's disease. These findings are in contrast with earlier studies, which reported impairment of proteasome activity in human Alzheimer's disease tissue and mouse models. Targeting the increased immunoproteasome activity with a specific inhibitor resulted in a decreased expression of inflammatory markers in ex vivo microglia. This may serve as a potential novel approach to modulate sustained neuroinflammation and glial dysfunction associated with Alzheimer's disease.