Neuroligin-3 and neuroligin-4X form nanoscopic clusters and regulate growth cone organization and size.

The cell-adhesion proteins neuroligin-3 and neuroligin-4X (NLGN3/4X) have well described roles in synapse formation. NLGN3/4X are also expressed highly during neurodevelopment. However, the role these proteins play during this period is unknown. Here we show that NLGN3/4X localized to the leading edge of growth cones where it promoted neuritogenesis in immature human neurons. Super-resolution microscopy revealed that NLGN3/4X clustering induced growth cone enlargement and influenced actin filament organization. Critically, these morphological effects were not induced by autism spectrum disorder (ASD)-associated NLGN3/4X variants. Finally, actin regulators p21-activated kinase 1 and cofilin were found to be activated by NLGN3/4X and involved in mediating the effects of these adhesion proteins on actin filaments, growth cones and neuritogenesis. These data reveal a novel role for NLGN3 and NLGN4X in the development of neuronal architecture, which may be altered in the presence of ASD-associated variants.

Brain-synthesized oestrogens regulate cortical migration in a sexually divergent manner.

Oestrogens play an important role in brain development where they have been implicated in controlling various cellular processes. Several lines of evidence have been presented showing that oestrogens can be synthesized locally within the brain. Studies have demonstrated that aromatase, the enzyme responsible for the conversion of androgens to oestrogens, is expressed during early development in both male and female cortices. Furthermore, 17ß-oestradiol has been measured in foetal brain tissue from multiple species. 17ß-oestradiol regulates neural progenitor proliferation as well as the development of early neuronal morphology. However, what role locally derived oestrogens play in regulating cortical migration and, moreover, whether these effects are the same in males and females are unknown. Here, we investigated the impact of knockdown expression of Cyp19a1, which encodes aromatase, between embryonic day (E) 14.5 and postnatal day 0 (P0) had on neural migration within the cortex. Aromatase was expressed in the developing cortex of both sexes, but at significantly higher levels in male than female mice. Under basal conditions, no obvious differences in cortical migration between male and female mice were observed. However, knockdown of Cyp19a1 resulted in an increase in cells within the cortical plate, and a concurrent decrease in the subventricular zone/ventricular zone in P0 male mice. Interestingly, the opposite effect was observed in females, who displayed a significant reduction in cells migrating to the cortical plate. Together, these findings indicate that brain-derived oestrogens regulate radial migration through distinct mechanisms in males and females.

Amyloid ß synaptotoxicity is Wnt-PCP dependent and blocked by fasudil.

INTRODUCTION: Synapse loss is the structural correlate of the cognitive decline indicative of dementia. In the brains of Alzheimer's disease sufferers, amyloid ß (Aß) peptides aggregate to form senile plaques but as soluble peptides are toxic to synapses. We previously demonstrated that Aß induces Dickkopf-1 (Dkk1), which in turn activates the Wnt-planar cell polarity (Wnt-PCP) pathway to drive tau pathology and neuronal death. METHODS: We compared the effects of Aß and of Dkk1 on synapse morphology and memory impairment while inhibiting or silencing key elements of the Wnt-PCP pathway. RESULTS: We demonstrate that Aß synaptotoxicity is also Dkk1 and Wnt-PCP dependent, mediated by the arm of Wnt-PCP regulating actin cytoskeletal dynamics via Daam1, RhoA and ROCK, and can be blocked by the drug fasudil. DISCUSSION: Our data add to the importance of aberrant Wnt signaling in Alzheimer's disease neuropathology and indicate that fasudil could be repurposed as a treatment for the disease.

Estradiol reverses excitatory synapse loss in a cellular model of neuropsychiatric disorders.

Loss of glutamatergic synapses is thought to be a key cellular pathology associated with neuropsychiatric disorders including schizophrenia (SCZ) and major depressive disorder (MDD). Genetic and cellular studies of SCZ and MDD using in vivo and in vitro systems have supported a key role for dysfunction of excitatory synapses in the pathophysiology of these disorders. Recent clinical studies have demonstrated that the estrogen, 17ß-estradiol can ameliorate many of the symptoms experienced by patients. Yet, to date, our understanding of how 17ß-estradiol exerted these beneficial effects is limited. In this study, we have tested the hypothesis that 17ß-estradiol can restore dendritic spine number in a cellular model that recapitulates the loss of synapses associated with SCZ and MDD. Ectopic expression of wildtype, mutant or shRNA-mediated knockdown of Disrupted in Schizophrenia 1 (DISC1) reduced dendritic spine density in primary cortical neurons. Acute or chronic treatment with 17ß-estradiol increased spine density to control levels in neurons with altered DISC1 levels. In addition, 17ß-estradiol reduced the extent to which ectopic wildtype and mutant DISC1 aggregated. Furthermore, 17ß-estradiol also caused the enrichment of synaptic proteins at synapses and increased the number of dendritic spines containing PSD-95 or that overlapped with the pre-synaptic marker bassoon. Taken together, our data indicates that estrogens can restore lost excitatory synapses caused by altered DISC1 expression, potentially through the trafficking of DISC1 and its interacting partners. These data highlight the possibility that estrogens exert their beneficial effects in SCZ and MDD in part by modulating dendritic spine number.

Cyto-nuclear shuttling of afadin is required for rapid estradiol-mediated modifications of histone H3.

Estrogens have been shown to rapidly regulate local signalling at synapses and within the nucleus. The result of these signalling events is to rapidly modulate synapse structure and function, as well as epigenetic mechanisms including histone modifications. Ultimately these mechanisms are thought to contribute to long-lasting changes in neural circuitry, and thus influence cognitive functions such as learning and memory. However, the mechanisms by which estrogen-mediated local synaptic and nuclear signalling events are coordinated are not well understood. In this study we have found that the scaffold protein afadin, (also known as AF-6), undergoes a bi-directional trafficking to both synaptic and nuclear compartment in response to acute 17ß-estradiol (estradiol) treatment, in mixed sex neuronal cultures derived from fetal cortex. Interestingly, nuclear accumulation of afadin was coincidental with an increase in the phosphorylation of histone H3 at serine 10 (H3S10p). This epigenetic modification is associated with the remodeling of chromatin into an open euchromatin state, allowing for transcriptional activation and related learning and memory processes. Critically, the cyto-nuclear trafficking of afadin was required for estradiol-dependent H3S10p. We further determined that nuclear accumulation of afadin is sufficient to induce phosphorylation of the mitogentic kinases ERK1/2 (pERK1/2) within the nucleus. Moreover, nuclear pERK1/2 was required for estradiol-dependent H3S10p. Taken together, we propose a model whereby estradiol induces the bi-directional trafficking of afadin to synaptic and nuclear sub-compartments. Within the nucleus, afadin is required for increased pERK1/2 which in turn is required for H3S10p. Therefore this represents a mechanism through which estrogens may be able to coordinate both synaptic and nucleosomal events within the same neuronal population.

Estradiol and the Development of the Cerebral Cortex: An Unexpected Role?

The cerebral cortex undergoes rapid folding in an "inside-outside" manner during embryonic development resulting in the establishment of six discrete cortical layers. This unique cytoarchitecture occurs via the coordinated processes of neurogenesis and cell migration. In addition, these processes are fine-tuned by a number of extracellular cues, which exert their effects by regulating intracellular signaling pathways. Interestingly, multiple brain regions have been shown to develop in a sexually dimorphic manner. In many cases, estrogens have been demonstrated to play an integral role in mediating these sexual dimorphisms in both males and females. Indeed, 17ß-estradiol, the main biologically active estrogen, plays a critical organizational role during early brain development and has been shown to be pivotal in the sexually dimorphic development and regulation of the neural circuitry underlying sex-typical and socio-aggressive behaviors in males and females. However, whether and how estrogens, and 17ß-estradiol in particular, regulate the development of the cerebral cortex is less well understood. In this review, we outline the evidence that estrogens are not only present but are engaged and regulate molecular machinery required for the fine-tuning of processes central to the cortex. We discuss how estrogens are thought to regulate the function of key molecular players and signaling pathways involved in corticogenesis, and where possible, highlight if these processes are sexually dimorphic. Collectively, we hope this review highlights the need to consider how estrogens may influence the development of brain regions directly involved in the sex-typical and socio-aggressive behaviors as well as development of sexually dimorphic regions such as the cerebral cortex.

The Progestin Receptor Interactome in the Female Mouse Hypothalamus: Interactions with Synaptic Proteins Are Isoform Specific and Ligand Dependent.

Progestins bind to the progestin receptor (PR) isoforms, PR-A and PR-B, in brain to influence development, female reproduction, anxiety, and stress. Hormone-activated PRs associate with multiple proteins to form functional complexes. In the present study, proteins from female mouse hypothalamus that associate with PR were isolated using affinity pull-down assays with glutathione S-transferase-tagged mouse PR-A and PR-B. Using complementary proteomics approaches, reverse phase protein array (RPPA) and mass spectrometry, we identified hypothalamic proteins that interact with PR in a ligand-dependent and isoform-specific manner and were confirmed by Western blot. Synaptic proteins, including synapsin-I and synapsin-II, interacted with agonist-bound PR isoforms, suggesting that both isoforms function in synaptic plasticity. In further support, synaptogyrin-III and synapsin-III associated with PR-A and PR-B, respectively. PR also interacted with kinases, including c-Src, mTOR, and MAPK1, confirming phosphorylation as an integral process in rapid effects of PR in the brain. Consistent with a role in transcriptional regulation, PR associated with transcription factors and coactivators in a ligand-specific and isoform-dependent manner. Interestingly, both PR isoforms associated with a key regulator of energy homeostasis, FoxO1, suggesting a novel role for PR in energy metabolism. Because many identified proteins in this PR interactome are synaptic proteins, we tested the hypothesis that progestins function in synaptic plasticity. Indeed, progesterone enhanced synaptic density, by increasing synapsin-I-positive synapses, in rat primary cortical neuronal cultures. This novel combination of RPPA and mass spectrometry allowed identification of PR action in synaptic remodeling and energy homeostasis and reveals unique roles for progestins in brain function and disease.

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.

Rapid modulation of synaptogenesis and spinogenesis by 17ß-estradiol in primary cortical neurons.

In the mammalian forebrain, the majority of excitatory synapses occur on dendritic spines. Changes in the number of these structures is important for brain development, plasticity and the refinement of neuronal circuits. The formation of excitatory synapses involves the coordinated formation of dendritic spines and targeting of multi-protein complexes to nascent connections. Recent studies have demonstrated that the estrogen 17ß-estradiol (E2) can rapidly increase the number of dendritic spines, an effect consistent with the ability of E2 to rapidly influence cognitive function. However, the molecular composition of E2-induced spines and whether these protrusions form synaptic connections has not been fully elucidated. Moreover, which estrogen receptor(s) (ER) mediate these spine-morphogenic responses are not clear. Here, we report that acute E2 treatment results in the recruitment of postsynaptic density protein 95 (PSD-95) to novel dendritic spines. In addition neuroligin 1 (Nlg-1) and the NMDA receptor subunit GluN1 are recruited to nascent synapses in cortical neurons. The presence of these synaptic proteins at nascent synapses suggests that the machinery to allow pre- and post-synapses to form connections are present in E2-induced spines. We further demonstrate that E2 treatment results in the rapid and transient activation of extracellular signal-regulated kinase 1/2 (ERK1/2), Akt and the mammalian target of rapamycin (mTOR) signaling pathways. However, only ERK1/2 and Akt are required for E2-mediated spinogenesis. Using synthetic receptor modulators, we further demonstrate that activation of the estrogen receptor beta (ERß) but not alpha (ERα) mimics rapid E2-induced spinogenesis and synaptogenesis. Taken together these findings suggest that in primary cortical neurons, E2 signaling via ERß, but not through ERα, is capable of remodeling neuronal circuits by increasing the number of excitatory synapses.