Human cerebellar organoids with functional Purkinje cells.

Research on human cerebellar development and disease has been hampered by the need for a human cell-based system that recapitulates the human cerebellum's cellular diversity and functional features. Here, we report a human organoid model (human cerebellar organoids [hCerOs]) capable of developing the complex cellular diversity of the fetal cerebellum, including a human-specific rhombic lip progenitor population that have never been generated in vitro prior to this study. 2-month-old hCerOs form distinct cytoarchitectural features, including laminar organized layering, and create functional connections between inhibitory and excitatory neurons that display coordinated network activity. Long-term culture of hCerOs allows healthy survival and maturation of Purkinje cells that display molecular and electrophysiological hallmarks of their in vivo counterparts, addressing a long-standing challenge in the field. This study therefore provides a physiologically relevant, all-human model system to elucidate the cell-type-specific mechanisms governing cerebellar development and disease.

Defining the nature of human pluripotent stem cell-derived interneurons via single-cell analysis.

The specification of inhibitory neurons has been described for the mouse and human brain, and many studies have shown that pluripotent stem cells (PSCs) can be used to create interneurons in vitro. It is unclear whether in vitro methods to produce human interneurons generate all the subtypes found in brain, and how similar in vitro and in vivo interneurons are. We applied single-nuclei and single-cell transcriptomics to model interneuron development from human cortex and interneurons derived from PSCs. We provide a direct comparison of various in vitro interneuron derivation methods to determine the homogeneity achieved. We find that PSC-derived interneurons capture stages of development prior to mid-gestation, and represent a minority of potential subtypes found in brain. Comparison with those found in fetal or adult brain highlighted decreased expression of synapse-related genes. These analyses highlight the potential to tailor the method of generation to drive formation of particular subtypes.

Identification of neural oscillations and epileptiform changes in human brain organoids.

Brain organoids represent a powerful tool for studying human neurological diseases, particularly those that affect brain growth and structure. However, many diseases manifest with clear evidence of physiological and network abnormality in the absence of anatomical changes, raising the question of whether organoids possess sufficient neural network complexity to model these conditions. Here, we explore the network-level functions of brain organoids using calcium sensor imaging and extracellular recording approaches that together reveal the existence of complex network dynamics reminiscent of intact brain preparations. We demonstrate highly abnormal and epileptiform-like activity in organoids derived from induced pluripotent stem cells from individuals with Rett syndrome, accompanied by transcriptomic differences revealed by single-cell analyses. We also rescue key physiological activities with an unconventional neuroregulatory drug, pifithrin-α. Together, these findings provide an essential foundation for the utilization of brain organoids to study intact and disordered human brain network formation and illustrate their utility in therapeutic discovery.

Transient muscarinic and glutamatergic stimulation of neural stem cells triggers acute and persistent changes in differentiation.

While aberrant cell proliferation and differentiation may contribute to epileptogenesis, the mechanisms linking an initial epileptic insult to subsequent changes in cell fate remain elusive. Using both mouse and human iPSC-derived neural progenitor/stem cells (NPSCs), we found that a combined transient muscarinic and mGluR1 stimulation inhibited overall neurogenesis but enhanced NPSC differentiation into immature GABAergic cells. If treated NPSCs were further passaged, they retained a nearly identical phenotype upon differentiation. A similar profusion of immature GABAergic cells was seen in rats with pilocarpine-induced chronic epilepsy. Furthermore, live cell imaging revealed abnormal de-synchrony of Ca(++) transients and altered gap junction intercellular communication following combined muscarinic/glutamatergic stimulation, which was associated with either acute site-specific dephosphorylation of connexin 43 or a long-term enhancement of its degradation. Therefore, epileptogenic stimuli can trigger acute and persistent changes in cell fate by altering distinct mechanisms that function to maintain appropriate intercellular communication between coupled NPSCs.

Cooperativity and complementarity: synergies in non-classical and classical glucocorticoid signaling.

Glucocorticoids (GCs) are an ubiquitous class of steroid hormones that exert a wide array of physiological effects. Traditionally, GC action has been considered to primarily involve transcriptional effects following the binding of hormone to the glucocorticoid receptor (GR) and subsequent activation or repression of target genes. However, a number of findings suggest that cellular responses following GC exposure may be mediated by transcription-independent, or "non-classical," mechanisms. We have added to this growing body of work by recently uncovering a novel GC signaling pathway that operates through plasma membrane GRs to limit gap junction intercellular signaling and limit the proliferation of neural progenitor cells (NPCs). In this review, we highlight our current state of knowledge of non-classical GR signaling, in particular as it applies to neuronal function. Using NPCs as a cellular model, we speculate on the components of this non-classical pathway and the mechanisms whereby a number of cytoplasmic and nuclear signaling events may be integrated.

Nongenomic glucocorticoid receptor action regulates gap junction intercellular communication and neural progenitor cell proliferation.

Glucocorticoids (GCs) are used to treat pregnant women at risk for preterm delivery; however, prenatal exposure to GCs may trigger adverse neurological side effects due to reduced neural progenitor cell (NPC) proliferation. Whereas many established cell-cycle regulators impact NPC proliferation, other signaling molecules, such as the gap junction protein connexin-43 (Cx43), also influence proliferation. Gap junction intercellular communication (GJIC) is influenced by GCs in some cells, but such hormone effects have not been examined in coupled stem cells. We found that both continuous and transient exposure of embryonic day 14.5 mouse neurosphere cultures to dexamethasone (DEX) limits proliferation of coupled NPCs, which is manifested by both a reduction in S-phase progression and enhanced cell-cycle exit. A short (i.e., 1-h) DEX treatment also reduced GJIC as measured by live-cell fluorescence recovery after photobleaching, and altered the synchrony of spontaneous calcium transients in coupled NPCs. GC effects on GJIC in NPCs are transcription-independent and mediated through plasma membrane glucocorticoid receptors (GRs). This nongenomic pathway operates through lipid raft-associated GRs via a site-specific, MAPK-dependent phosphorylation of Cx43, which is linked to GR via caveolin-1 (Cav-1) and c-src. Cav-1 is essential for this nongenomic action of GR, as DEX effects on GJIC, Cx43 phosphorylation, and MAPK activation are not observed in Cav-1 knockout NPCs. As transient pharmacologic inhibition of GJIC triggers reduced S-phase progression but not enhanced cell-cycle exit, the nongenomic GR signaling pathway may operate via distinct downstream effectors to alter the proliferative capacity of NPCs.

The dopamine and cAMP regulated phosphoprotein, 32 kDa (DARPP-32) signaling pathway: a novel therapeutic target in traumatic brain injury.

Traumatic brain injury (TBI) causes persistent neurologic deficits. Current therapies, predominantly focused upon cortical and hippocampal cellular survival, have limited benefit on cognitive outcomes. Striatal damage is associated with deficits in executive function, learning, and memory. Dopamine and cAMP regulated phosphoprotein 32 (DARPP-32) is expressed within striatal medium spiny neurons and regulates striatal function. We found that controlled cortical impact injury in rats produces a chronic decrease in DARPP-32 phosphorylation at threonine-34 and an increase in protein phosphatase-1 activity. There is no effect of injury on threonine-75 phosphorylation or on DARPP-32 protein. Amantadine, shown to be efficacious in treating post-TBI cognitive deficits, given daily for two weeks is able to restore the loss of DARPP-32 phosphorylation and reduce protein phosphatase-1 activity. Amantadine also decreases the phosphorylation of threonine-75 consistent with activity as a partial N-methyl-D-aspartate (NMDA) receptor antagonist and partial dopamine agonist. These data demonstrate that targeting the DARPP-32 signaling cascade represents a promising novel therapeutic approach in the treatment of persistent deficits following a TBI.

Selective inhibition of mitogen-activated protein kinase phosphatases by zinc accounts for extracellular signal-regulated kinase 1/2-dependent oxidative neuronal cell death.

Oxidative stress induced by glutathione depletion in the mouse HT22 neuroblastoma cell line and embryonic rat immature cortical neurons causes a delayed, sustained activation of extracellular signal-regulated kinase (ERK) 1/2, which is required for cell death. This sustained activation of ERK1/2 is mediated primarily by a selective inhibition of distinct ERK1/2-directed phosphatases either by enhanced degradation (i.e., for mitogen-activated protein kinase phosphatase-1) or as shown here by reductions in enzymatic activity (i.e., for protein phosphatase type 2A). The inhibition of ERK1/2 phosphatases in HT22 cells and immature neurons subjected to glutathione depletion results from oxidative stress because phosphatase activity is restored in cells treated with the antioxidant butylated hydroxyanisole. This leads to reduced ERK1/2 activation and neuroprotection. Furthermore, an increase in free intracellular zinc that accompanies glutathione-induced oxidative stress in HT22 cells and immature neurons contributes to selective inhibition of ERK1/2 phosphatase activity and cell death. Finally, ERK1/2 also functions to maintain elevated levels of zinc. Thus, the elevation of intracellular zinc within neurons subjected to oxidative stress can trigger a robust positive feedback loop operating through activated ERK1/2 that rapidly sets into motion a zinc-dependent pathway of cell death.

Induction of an anti-inflammatory cytokine, IL-10, in dendritic cells after toll-like receptor signaling.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that modulates innate and adaptive immunity. IL-10 transcripts and the protein were induced in murine bone marrow-derived dendritic cells (BMDCs) after toll-like receptor (TLR) stimulation. IL-10 induction was TLR ligand selective, in that CpG DNA, imidazoquinolin, peptidoglycan, and zymosan but not lipopolysaccharide (LPS) and poly I:C led to IL-10 production. IL-10 induction was, however, completely absent in MyD88(/) DCs that lacked a TLR adaptor showing that IL-10 induction depends on TLR signaling. Kinetic analysis of IL-10 induction by CpG and imidazoquinolin revealed a prolonged lag phase prior to a measurable rise in transcript levels, which peaked at 12-24 h after stimulation. Stat3, implicated in IL-10 gene transcription, was also induced after TLR stimulation with the kinetics similar to those of IL-10 induction. Further, Stat3 was phosphorylated and bound to the IL-10 promoter in TLR-stimulated DCs. Supporting a link with IL-10 induction, STAT3 induction was absent in MyD88(/) DCs. These data suggest a two-step model where the initial TLR signaling induced proinflammatory cytokines, which then activated Stat3, leading to the induction of IL-10. TLR-stimulated IL-10 production may regulate DC maturation steps, thereby influencing the ensuing immune responses.