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

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

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

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

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

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 crucia

Engineered cell culture microenvironments for mechanobiology studies of brain neural cells

The biomechanical properties of the brain microenvironment, which is composed of different neural cell types, the extracellular matrix, and blood vessels, are critical for normal brain development and neural functioning. Stiffness, viscoelasticity and spatial organization of brain tissue modulate proliferation, migration, differentiation, and cell function. However, the mechanical aspects of the neural microenvironment are largely ignored in current cell culture systems. Considering the high promises of human induced pluripotent stem cell- (iPSC-) based models for disease modelling and new treatment development, and in light of the physiological relevance of neuromechanobiological features, applications of in vitro engineered neuronal microenvironments should be explored thoroughly to develop more representative in vitro brain models. In this context, recently developed biomaterials in combination with micro- and nanofabrication techniques 1) allow investigating how mechanical properties affect neural cell development and functioning; 2) enable optimal cell microenvironment engineering strategies to advance neural cell models; and 3) provide a quantitative tool to assess changes in the neuromechanobiological properties of the brain microenvironment induced by pathology. In this review, we discuss the biological and engineering aspects involved in studying neuromechanobiology within scaffold-free and scaffold-based 2D and 3D iPSC-based brain models and approaches employing primary lineages (neural/glial), cell lines and other stem cells. Finally, we discuss future experimental directions of engineered microenvironments in neuroscience

Axonal abnormalities in vanishing white matter

ObjectiveWe aimed to study the occurrence and development of axonal pathology and the influence of astrocytes in vanishing white matter. MethodsAxons and myelin were analyzed using electron microscopy and immunohistochemistry on Eif2b4 and Eif2b5 single- and double-mutant mice and patient brain tissue. In addition, astrocyte-forebrain co-culture studies were performed. ResultsIn the corpus callosum of Eif2b5-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 Eif2b5-mutant mice. In more severely affected Eif2b4-Eif2b5 double-mutants, similar abnormalities were already present at 4 months, while in milder affected Eif2b4 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 Eif2b5-mutant mice. Co-cultures showed that Eif2b5 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. InterpretationIn 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

Neuron-glia interactions increase neuronal phenotypes in tuberous sclerosis complex patient iPSC-derived models

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

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

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

Simplified 3D protocol capable of generating early cortical neuroepithelium

Here,we report a 3Dcerebellar differentiation protocolwith 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 granulecells).Follow-uptestsindicatedthatadditionofFGFs, 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 costefficientmethod for 3D culture, whether to research development of the early cerebellar neuroepithelium, a base to generate mature cortical structures,or tooptimizeminimal-factorprotocols forother brainregions

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

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