Human cortical spheroids with a high diversity of innately developing brain cell types.

BACKGROUND: Three-dimensional (3D) human brain spheroids are instrumental to study central nervous system (CNS) development and (dys)function. Yet, in current brain spheroid models the limited variety of cell types hampers an integrated exploration of CNS (disease) mechanisms. METHODS: Here we report a 5-month culture protocol that reproducibly generates H9 embryonic stem cell-derived human cortical spheroids (hCSs) with a large cell-type variety. RESULTS: We established the presence of not only neuroectoderm-derived neural progenitor populations, mature excitatory and inhibitory neurons, astrocytes and oligodendrocyte (precursor) cells, but also mesoderm-derived microglia and endothelial cell populations in the hCSs via RNA-sequencing, qPCR, immunocytochemistry and transmission electron microscopy. Transcriptomic analysis revealed resemblance between the 5-months-old hCSs and dorsal frontal rather than inferior regions of human fetal brains of 19-26 weeks of gestational age. Pro-inflammatory stimulation of the generated hCSs induced a neuroinflammatory response, offering a proof-of-principle of the applicability of the spheroids. CONCLUSIONS: Our protocol provides a 3D human brain cell model containing a wide variety of innately developing neuroectoderm- as well as mesoderm-derived cell types, furnishing a versatile platform for comprehensive examination of intercellular CNS communication and neurological disease mechanisms.

Molecular Signature of Neuroinflammation Induced in Cytokine-Stimulated Human Cortical Spheroids.

Crucial in the pathogenesis of neurodegenerative diseases is the process of neuroinflammation that is often linked to the pro-inflammatory cytokines Tumor necrosis factor alpha (TNFα) and Interleukin-1beta (IL-1ß). Human cortical spheroids (hCSs) constitute a valuable tool to study the molecular mechanisms underlying neurological diseases in a complex three-dimensional context. We recently designed a protocol to generate hCSs comprising all major brain cell types. Here we stimulate these hCSs for three time periods with TNFα and with IL-1ß. Transcriptomic analysis reveals that the main process induced in the TNFα- as well as in the IL-1ß-stimulated hCSs is neuroinflammation. Central in the neuroinflammatory response are endothelial cells, microglia and astrocytes, and dysregulated genes encoding cytokines, chemokines and their receptors, and downstream NFκB- and STAT-pathway components. Furthermore, we observe sets of neuroinflammation-related genes that are specifically modulated in the TNFα-stimulated and in the IL-1ß-stimulated hCSs. Together, our results help to molecularly understand human neuroinflammation and thus a key mechanism of neurodegeneration.

Key role for lipids in cognitive symptoms of schizophrenia.

Schizophrenia (SZ) is a psychiatric disorder with a convoluted etiology that includes cognitive symptoms, which arise from among others a dysfunctional dorsolateral prefrontal cortex (dlPFC). In our search for the molecular underpinnings of the cognitive deficits in SZ, we here performed RNA sequencing of gray matter from the dlPFC of SZ patients and controls. We found that the differentially expressed RNAs were enriched for mRNAs involved in the Liver X Receptor/Retinoid X Receptor (LXR/RXR) lipid metabolism pathway. Components of the LXR/RXR pathway were upregulated in gray matter but not in white matter of SZ dlPFC. Intriguingly, an analysis for shared genetic etiology, using two SZ genome-wide association studies (GWASs) and GWAS data for 514 metabolites, revealed genetic overlap between SZ and acylcarnitines, VLDL lipids, and fatty acid metabolites, which are all linked to the LXR/RXR signaling pathway. Furthermore, analysis of structural T1-weighted magnetic resonance imaging in combination with cognitive behavioral data showed that the lipid content of dlPFC gray matter is lower in SZ patients than in controls and correlates with a tendency towards reduced accuracy in the dlPFC-dependent task-switching test. We conclude that aberrations in LXR/RXR-regulated lipid metabolism lead to a decreased lipid content in SZ dlPFC that correlates with reduced cognitive performance.

Reappraisal of Human HOG and MO3.13 Cell Lines as a Model to Study Oligodendrocyte Functioning.

Myelination of neuronal axons is essential for proper brain functioning and requires mature myelinating oligodendrocytes (myOLs). The human OL cell lines HOG and MO3.13 have been widely used as in vitro models to study OL (dys) functioning. Here we applied a number of protocols aimed at differentiating HOG and MO3.13 cells into myOLs. However, none of the differentiation protocols led to increased expression of terminal OL differentiation or myelin-sheath formation markers. Surprisingly, the applied protocols did cause changes in the expression of markers for early OLs, neurons, astrocytes and Schwann cells. Furthermore, we noticed that mRNA expression levels in HOG and MO3.13 cells may be affected by the density of the cultured cells. Finally, HOG and MO3.13 co-cultured with human neuronal SH-SY5Y cells did not show myelin formation under several pro-OL-differentiation and pro-myelinating conditions. Together, our results illustrate the difficulty of inducing maturation of HOG and MO3.13 cells into myOLs, implying that these oligodendrocytic cell lines may not represent an appropriate model to study the (dys)functioning of human (my)OLs and OL-linked disease mechanisms.

Leptin signaling in GABA neurons, but not glutamate neurons, is required for reproductive function.

The adipocyte-derived hormone leptin acts in the brain to modulate the central driver of fertility: the gonadotropin releasing hormone (GnRH) neuronal system. This effect is indirect, as GnRH neurons do not express leptin receptors (LEPRs). Here we test whether GABAergic or glutamatergic neurons provide the intermediate pathway between the site of leptin action and the GnRH neurons. Leptin receptors were deleted from GABA and glutamate neurons using Cre-Lox transgenics, and the downstream effects on puberty onset and reproduction were examined. Both mouse lines displayed the expected increase in body weight and region-specific loss of leptin signaling in the hypothalamus. The GABA neuron-specific LEPR knock-out females and males showed significantly delayed puberty onset. Adult fertility observations revealed that these knock-out animals have decreased fecundity. In contrast, glutamate neuron-specific LEPR knock-out mice displayed normal fertility. Assessment of the estrogenic hypothalamic-pituitary-gonadal axis regulation in females showed that leptin action on GABA neurons is not necessary for estradiol-mediated suppression of tonic luteinizing hormone secretion (an indirect measure of GnRH neuron activity) but is required for regulation of a full preovulatory-like luteinizing hormone surge. In conclusion, leptin signaling in GABAergic (but not glutamatergic neurons) plays a critical role in the timing of puberty onset and is involved in fertility regulation throughout adulthood in both sexes. These results form an important step in explaining the role of central leptin signaling in the reproductive system. Limiting the leptin-to-GnRH mediators to GABAergic cells will enable future research to focus on a few specific types of neurons.

Neither signal transducer and activator of transcription 3 (STAT3) or STAT5 signaling pathways are required for leptin's effects on fertility in mice.

The hormone leptin is critical for the regulation of energy balance and fertility. The long-form leptin receptor (LepR) regulates multiple intracellular signaling cascades, including the classic Janus kinase-signal transducer and activator of transcription (STAT) pathways. Previous studies have shown that deletion of STAT3 or the closely related STAT5 from the brain results in an obese phenotype, but their roles in fertility regulation are not clear. This study tested whether STAT3 and STAT5 pathways of leptin signaling are required for fertility, and whether absence of one pathway might be compensated for by the other in a redundant manner. A Cre-loxP approach was used to generate 3 models of male and female transgenic mice with LepR-specific deletion of STAT3, STAT5, or both STAT3 and STAT5. Body weight, puberty onset, estrous cyclicity, and fertility were measured in all knockout (KO) mice and their control littermates. Knocking out STAT3 or both STAT3 and 5 from LepR expressing cells, but not STAT5 alone, led to significant increase in body weight. All STAT3 and STAT5 single KO mice exhibited normal puberty onset and subsequent fertility compared to their control littermates. Surprisingly, all STAT3 and STAT5 double KO mice also exhibited normal puberty onset, estrous cyclicity, and fertility, although they had severely disrupted body weight regulation. These results suggest that, although STAT3 signaling is crucial for body weight regulation, neither STAT3 nor STAT5 is required for the regulation of fertility by leptin. It remains to be determined what other signaling molecules mediate this effect of leptin, and whether they interact in a redundant manner.