Tracking neuronal motility in live murine retinal explants.

The developing retina undergoes dynamic organizational changes involving significant intra-retinal motility of the encompassing cells. Here, we present a protocol for tracking retinal cell motility in live explanted mouse retinae. Although originally applied to rod and cone photoreceptors, this strategy is applicable to any fluorescently labeled cell in mouse retinae and other similar experimental retinal models. Careful tissue handling is critical for the successful acquisition of high-quality live imaging data. Further instructions for semi-automated in silico data handling are provided. For complete details on the use and execution of this protocol, please refer to Aghaizu et al. (2021).

Repeated nuclear translocations underlie photoreceptor positioning and lamination of the outer nuclear layer in the mammalian retina.

In development, almost all stratified neurons must migrate from their birthplace to the appropriate neural layer. Photoreceptors reside in the most apical layer of the retina, near their place of birth. Whether photoreceptors require migratory events for fine-positioning and/or retention within this layer is not well understood. Here, we show that photoreceptor nuclei of the developing mouse retina cyclically exhibit rapid, dynein-1-dependent translocation toward the apical surface, before moving more slowly in the basal direction, likely due to passive displacement by neighboring retinal nuclei. Attenuating dynein 1 function in rod photoreceptors results in their ectopic basal displacement into the outer plexiform layer and inner nuclear layer. Synapse formation is also compromised in these displaced cells. We propose that repeated, apically directed nuclear translocation events are necessary to ensure retention of post-mitotic photoreceptors within the emerging outer nuclear layer during retinogenesis, which is critical for correct neuronal lamination.

Dopamine-induced interactions of female mouse hypothalamic proteins with progestin receptor-A in the absence of hormone.

Neural progestin receptors (PR) function in reproduction, neural development, neuroprotection, learning, memory and the anxiety response. In the absence of progestins, PR can be activated by dopamine (DA) in the rodent hypothalamus to elicit female sexual behaviour. The present study investigated mechanisms of DA activation of PR by testing the hypothesis that proteins from DA-treated hypothalami interact with PR in the absence of progestins. Ovariectomised, oestradiol-primed mice were infused with a D1-receptor agonist, SKF38393 (SKF), into the third ventricle 30 minutes prior to death. Proteins from SKF-treated hypothalami were pulled-down with glutathione S-transferase-tagged mouse PR-A or PR-B and the interactomes were analysed by mass spectrometry. The largest functional group to interact with PR-A in a DA-dependent manner was synaptic proteins. To test the hypothesis that DA activation of PR regulates synaptic proteins, we developed oestradiol-induced PR-expressing hypothalamic-like neurones derived from human-induced pluripotent stem cells (hiPSCs). Similar to progesterone (P4), SKF treatment of hiPSCs increased synapsin1/2 expression. This SKF-dependent effect was blocked by the PR antagonist RU486, suggesting that PR are necessary for this DA-induced increase. The second largest DA-dependent PR-A protein interactome comprised metabolic regulators involved in glucose metabolism, lipid synthesis and mitochondrial energy production. Interestingly, hypothalamic proteins interacted with PR-A, but not PR-B, in an SKF-dependent manner, suggesting that DA promotes the interaction of multiple hypothalamic proteins with PR-A. These in vivo and in vitro results indicate novel mechanisms by which DA can differentially activate PR isoforms in the absence of P4 and provide a better understanding of ligand-independent PR activation in reproductive, metabolic and mental health disorders in women.

Interferon-γ signaling in human iPSC-derived neurons recapitulates neurodevelopmental disorder phenotypes.

Maternal immune activation increases the risk of neurodevelopmental disorders. Elevated cytokines, such as interferon-γ (IFN-γ), in offspring's brains play a central role. IFN-γ activates an antiviral cellular state, limiting viral entry and replication. Moreover, IFN-γ is implicated in brain development. We tested the hypothesis that IFN-γ signaling contributes to molecular and cellular phenotypes associated with neurodevelopmental disorders. Transient IFN-γ treatment of neural progenitors derived from human induced pluripotent stem cells increased neurite outgrowth. RNA sequencing analysis revealed that major histocompatibility complex class I (MHCI) genes were persistently up-regulated through neuronal differentiation-an effect that was mediated by IFN-γ-induced promyelocytic leukemia protein (PML) nuclear bodies. Critically, IFN-γ-induced neurite outgrowth required both PML and MHCI. We also found evidence that IFN-γ disproportionately altered the expression of genes associated with schizophrenia and autism, suggesting convergence between genetic and environmental risk factors. Together, these data implicate IFN-γ signaling in neurodevelopmental disorder etiology.

Δ9-tetrahydrocannabinol and 2-AG decreases neurite outgrowth and differentially affects ERK1/2 and Akt signaling in hiPSC-derived cortical neurons.

Endocannabinoids regulate different aspects of neurodevelopment. In utero exposure to the exogenous psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC), has been linked with abnormal cortical development in animal models. However, much less is known about the actions of endocannabinoids in human neurons. Here we investigated the effect of the endocannabinoid 2-arachidonoyl glycerol (2AG) and Δ9-THC on the development of neuronal morphology and activation of signaling kinases, in cortical neurons derived from human induced pluripotent stem cells (hiPSCs). Our data indicate that the cannabinoid type 1 receptor (CB1R), but not the cannabinoid 2 receptor (CB2R), GPR55 or TRPV1 receptors, is expressed in young, immature hiPSC-derived cortical neurons. Consistent with previous reports, 2AG and Δ9-THC negatively regulated neurite outgrowth. Interestingly, acute exposure to both 2AG and Δ9-THC inhibited phosphorylation of serine/threonine kinase extracellular signal-regulated protein kinases (ERK1/2), whereas Δ9-THC also reduced phosphorylation of Akt (aka PKB). Moreover, the CB1R inverse agonist SR 141716A attenuated the decrease in neurite outgrowth and ERK1/2 phosphorylation induced by 2AG and Δ9-THC. Taken together, our data suggest that hiPSC-derived cortical neurons express CB1Rs and are responsive to exogenous cannabinoids. Thus, hiPSC-neurons may represent a good cellular model for investigating the role of the endocannabinoid system in regulating cellular processes in developing human neurons.

Psychosis Risk Candidate ZNF804A Localizes to Synapses and Regulates Neurite Formation and Dendritic Spine Structure.

BACKGROUND: Variation in the gene encoding zinc finger binding protein 804A (ZNF804A) is associated with schizophrenia and bipolar disorder. Evidence suggests that ZNF804A is a regulator of gene transcription and is present in nuclear and extranuclear compartments. However, a detailed examination of ZNF804A distribution and its neuronal functions has yet to be performed. METHODS: The localization of ZNF804A protein was examined in neurons derived from human neural progenitor cells, human induced pluripotent stem cells, or in primary rat cortical neurons. In addition, small interfering RNA-mediated knockdown of ZNF804A was conducted to determine its role in neurite formation, maintenance of dendritic spine morphology, and responses to activity-dependent stimulations. RESULTS: Endogenous ZNF804A protein localized to somatodendritic compartments and colocalized with the putative synaptic markers in young neurons derived from human neural progenitor cells and human induced pluripotent stem cells. In mature rat neurons, Zfp804A, the homolog of ZNF804A, was present in a subset of dendritic spines and colocalized with synaptic proteins in specific nanodomains, as determined by super-resolution microscopy. Interestingly, knockdown of ZNF804A attenuated neurite outgrowth in young neurons, an effect potentially mediated by reduced neuroligin-4 expression. Furthermore, knockdown of ZNF804A in mature neurons resulted in the loss of dendritic spine density and impaired responses to activity-dependent stimulation. CONCLUSIONS: These data reveal a novel subcellular distribution for ZNF804A within somatodendritic compartments and a nanoscopic organization at excitatory synapses. Moreover, our results suggest that ZNF804A plays an active role in neurite formation, maintenance of dendritic spines, and activity-dependent structural plasticity.

Utilizing induced pluripotent stem cells (iPSCs) to understand the actions of estrogens in human neurons.

This article is part of a Special Issue "Estradiol and Cognition". Over recent years tremendous progress has been made towards understanding the molecular and cellular mechanism by which estrogens exert enhancing effects on cognition, and how they act as a neuroprotective or neurotrophic agent in disease. Currently, much of this work has been carried out in animal models with only a limited number of studies using native human tissue or cells. Recent advances in stem cell technology now make it possible to reprogram somatic cells from humans into induced pluripotent stem cells (iPSCs), which can subsequently be differentiated into neurons of specific lineages. Importantly, the reprogramming of cells allows for the generation of iPSCs that retain the genetic "makeup" of the donor. Therefore, it is possible to generate iPSC-derived neurons from patients diagnosed with specific diseases, that harbor the complex genetic background associated with the disorder. Here, we review the iPSC technology and how it's currently being used to model neural development and neurological diseases. Furthermore, we explore whether this cellular system could be used to understand the role of estrogens in human neurons, and present preliminary data in support of this. We further suggest that the use of iPSC technology offers a novel system to not only further understand estrogens' effects in human cells, but also to investigate the mechanism by which estrogens are beneficial in disease. Developing a greater understanding of these mechanisms in native human cells will also aid in the development of safer and more effective estrogen-based therapeutics.

Migration, integration and maturation of photoreceptor precursors following transplantation in the mouse retina.

Retinal degeneration leading to loss of photoreceptors is a major cause of untreatable blindness. Recent research has yielded definitive evidence for restoration of vision following the transplantation of rod photoreceptors in murine models of blindness, while advances in stem cell biology have enabled the generation of transplantable photoreceptors from embryonic stem cells. Importantly, the amount of visual function restored is dependent upon the number of photoreceptors that migrate correctly into the recipient retina. The developmental stage of the donor cells is important for their ability to migrate; they must be immature photoreceptor precursors. Little is known about how and when donor cell migration, integration, and maturation occurs. Here, we have performed a comprehensive histological analysis of the 6-week period following rod transplantation in mice. Donor cells migrate predominately as single entities during the first week undergoing a stereotyped sequence of morphological changes in their translocation from the site of transplantation, through the interphotoreceptor matrix and into the recipient retina. This includes initial polarization toward the outer nuclear layer (ONL), followed by formation of an apical attachment and rudimentary segment during migration into the ONL. Strikingly, acquisition of a nuclear architecture typical of mature rods was accelerated compared with normal development and a feature of migrating cells. Once within the ONL, precursors formed synaptic-like structures and outer segments in accordance with normal maturation. The restoration of visual function mediated by transplanted photoreceptors correlated with the later expression of rod α-transducin, achieving maximal function by 5 weeks.

Repair of the degenerate retina by photoreceptor transplantation.

Despite different aetiologies, age-related macular degeneration and most inherited retinal disorders culminate in the same final common pathway, the loss of photoreceptors. There are few treatments and none reverse the loss of vision. Photoreceptor replacement by transplantation is proposed as a broad treatment strategy applicable to all degenerations. Recently, we demonstrated restoration of vision following rod-photoreceptor transplantation into a mouse model of stationary night-blindness, raising the critical question of whether photoreceptor replacement is equally effective in different types and stages of degeneration. We present a comprehensive assessment of rod-photoreceptor transplantation across six murine models of inherited photoreceptor degeneration. Transplantation is feasible in all models examined but disease type has a major impact on outcome, as assessed both by the morphology and number of integrated rod-photoreceptors. Integration can increase (Prph2(+/Δ307)), decrease (Crb1(rd8/rd8), Gnat1(-/-), Rho(-/-)), or remain constant (PDE6ß(rd1/rd1), Prph2(rd2/rd2)) with disease progression, depending upon the gene defect, with no correlation with severity. Robust integration is possible even in late-stage disease. Glial scarring and outer limiting membrane integrity, features that change with degeneration, significantly affect transplanted photoreceptor integration. Combined breakdown of these barriers markedly increases integration in a model with an intact outer limiting membrane, strong gliotic response, and otherwise poor transplantation outcome (Rho(-/-)), leading to an eightfold increase in integration and restoration of visual function. Thus, it is possible to achieve robust integration across a broad range of inherited retinopathies. Moreover, transplantation outcome can be improved by administering appropriate, tailored manipulations of the recipient environment.