Cannabidiol inhibits neuroinflammatory responses and circuit-associated synaptic loss following damage to a songbird vocal pre-motor cortical-like region.

The non-euphorigenic phytocannabinoid cannabidiol (CBD) has been used successfully to treat childhood-onset epilepsies. These conditions are associated with developmental delays that often include vocal learning. Zebra finch song, like language, is a complex behavior learned during a sensitive period of development. Song quality is maintained through continuous sensorimotor refinement involving circuits that control learning and production. Within the vocal motor circuit, HVC is a cortical-like region that when partially lesioned temporarily disrupts song structure. We previously found CBD (10 mg/kg/day) improves post-lesion vocal recovery. The present studies were done to begin to understand mechanisms possibly responsible for CBD vocal protection. We found CBD markedly reduced expression of inflammatory mediators and oxidative stress markers. These effects were associated with regionally-reduced expression of the microglial marker TMEM119. As microglia are key regulators of synaptic reorganization, we measured synapse densities, finding significant lesion-induced circuit-wide decreases that were largely reversed by CBD. Synaptic protection was accompanied by NRF2 activation and BDNF/ARC/ARG3.1/MSK1 expression implicating mechanisms important to song circuit node mitigation of oxidative stress and promotion of synaptic homeostasis. Our findings demonstrate that CBD promotes an array of neuroprotective processes consistent with modulation of multiple cell signaling systems, and suggest these mechanisms are important to post-lesion recovery of a complex learned behavior.

Shared mechanisms of neural circuit disruption in tuberous sclerosis across lifespan: Bridging neurodevelopmental and neurodegenerative pathology.

Tuberous Sclerosis (TS) is a rare genetic disorder manifesting with multiple benign tumors impacting the function of vital organs. In TS patients, dominant negative mutations in TSC1 or TSC2 increase mTORC1 activity. Increased mTORC1 activity drives tumor formation, but also severely impacts central nervous system function, resulting in infantile seizures, intractable epilepsy, and TS-associated neuropsychiatric disorders, including autism, attention deficits, intellectual disability, and mood disorders. More recently, TS has also been linked with frontotemporal dementia. In addition to TS, accumulating evidence implicates increased mTORC1 activity in the pathology of other neurodevelopmental and neurodegenerative disorders. Thus, TS provides a unique disease model to address whether developmental neural circuit abnormalities promote age-related neurodegeneration, while also providing insight into the therapeutic potential of mTORC1 inhibitors for both developing and degenerating neural circuits. In the following review, we explore the ability of both mouse and human brain organoid models to capture TS pathology, elucidate disease mechanisms, and shed light on how neurodevelopmental alterations may later contribute to age-related neurodegeneration.

The Applicability of Current Turbidimetric Approaches for Analyzing Fibrin Fibers and Other Filamentous Networks.

Turbidimetry is an experimental technique often used to study the structure of filamentous networks. To extract structural properties such as filament diameter from turbidimetric data, simplifications to light scattering theory must be employed. In this work, we evaluate the applicability of three commonly utilized turbidimetric analysis approaches, each using slightly different simplifications. We make a specific application towards analyzing fibrin fibers, which form the structural scaffold of blood clots, but the results are generalizable. Numerical simulations were utilized to assess the applicability of each approach across a range of fiber lengths and diameters. Simulation results indicated that all three turbidimetric approaches commonly underestimate fiber diameter, and that the "Carr-Hermans" approach, utilizing wavelengths in the range of 500−800 nm, provided <10% error for the largest number of diameter/length combinations. These theoretical results were confirmed, under select conditions, via the comparison of fiber diameters extracted from experimental turbidimetric data, with diameters obtained using super-resolution microscopy.

Effects of the Selective Serotonin Reuptake Inhibitor Fluoxetine on Developing Neural Circuits in a Model of the Human Fetal Cortex.

The developing prenatal brain is particularly susceptible to environmental disturbances. During prenatal brain development, synapses form between neurons, resulting in neural circuits that support complex cognitive functions. In utero exposure to environmental factors such as pharmaceuticals that alter the process of synapse formation increases the risk of neurodevelopmental abnormalities. However, there is a lack of research into how specific environmental factors directly impact the developing neural circuitry of the human brain. For example, selective serotonin reuptake inhibitors are commonly used throughout pregnancy to treat depression, yet their impact on the developing fetal brain remains unclear. Recently, human brain models have provided unprecedented access to the critical window of prenatal brain development. In the present study, we used human neurons and cortical spheroids to determine whether the selective serotonin reuptake inhibitor fluoxetine alters neurite and synapse formation and the development of spontaneous activity within neural circuits. We demonstrate that cortical spheroids express serotonin transporter, thus recapitulating the early developmental expression of serotonin transporter associated with cortical pyramidal neurons. Cortical spheroids also appropriately express serotonin receptors, such as synaptic 5-HT2A and glial 5-HT5A. To determine whether fluoxetine can affect developing neural circuits independent of serotonergic innervation from the dorsal and medial raphe nuclei, we treated cortical neurons and spheroids with fluoxetine. Fluoxetine alters neurite formation in a dose-dependent fashion. Intriguingly, in cortical spheroids, neither acute nor chronic fluoxetine significantly altered excitatory synapse formation. However, only acute, but not chronic fluoxetine exposure altered inhibitory synaptogenesis. Finally, fluoxetine reversibly suppresses neuronal activity in a dose-dependent manner. These results demonstrate that fluoxetine can acutely alter synaptic function in developing neural circuits, but the effects were not long-lasting. This work provides a foundation for future studies to combine serotonergic innervation with cortical spheroids and assess the contributions of fluoxetine-induced alterations in serotonin levels to brain development.

Synaptic Hyaluronan Synthesis and CD44-Mediated Signaling Coordinate Neural Circuit Development.

The hyaluronan-based extracellular matrix is expressed throughout nervous system development and is well-known for the formation of perineuronal nets around inhibitory interneurons. Since perineuronal nets form postnatally, the role of hyaluronan in the initial formation of neural circuits remains unclear. Neural circuits emerge from the coordinated electrochemical signaling of excitatory and inhibitory synapses. Hyaluronan localizes to the synaptic cleft of developing excitatory synapses in both human cortical spheroids and the neonatal mouse brain and is diminished in the adult mouse brain. Given this developmental-specific synaptic localization, we sought to determine the mechanisms that regulate hyaluronan synthesis and signaling during synapse formation. We demonstrate that hyaluronan synthase-2, HAS2, is sufficient to increase hyaluronan levels in developing neural circuits of human cortical spheroids. This increased hyaluronan production reduces excitatory synaptogenesis, promotes inhibitory synaptogenesis, and suppresses action potential formation. The hyaluronan receptor, CD44, promotes hyaluronan retention and suppresses excitatory synaptogenesis through regulation of RhoGTPase signaling. Our results reveal mechanisms of hyaluronan synthesis, retention, and signaling in developing neural circuits, shedding light on how disease-associated hyaluronan alterations can contribute to synaptic defects.

Acyl-CoA synthetase 6 is required for brain docosahexaenoic acid retention and neuroprotection during aging.

The omega-3 fatty acid docosahexaenoic acid (DHA) inversely relates to neurological impairments with aging; however, limited nondietary models manipulating brain DHA have hindered a direct linkage. We discovered that loss of long-chain acyl-CoA synthetase 6 in mice (Acsl6-/-) depletes brain membrane phospholipid DHA levels, independent of diet. Here, Acsl6-/- brains contained lower DHA compared with controls across the life span. The loss of DHA- and increased arachidonate-enriched phospholipids were visualized by MALDI imaging predominantly in neuron-rich regions where single-molecule RNA in situ hybridization localized Acsl6 to neurons. ACSL6 is also astrocytic; however, we found that astrocyte-specific ACSL6 depletion did not alter membrane DHA because astrocytes express a non-DHA-preferring ACSL6 variant. Across the life span, Acsl6-/- mice exhibited hyperlocomotion, impairments in working spatial memory, and increased cholesterol biosynthesis genes. Aging caused Acsl6-/- brains to decrease the expression of membrane, bioenergetic, ribosomal, and synaptic genes and increase the expression of immune response genes. With age, the Acsl6-/- cerebellum became inflamed and gliotic. Together, our findings suggest that ACSL6 promotes membrane DHA enrichment in neurons, but not in astrocytes, and is important for neuronal DHA levels across the life span. The loss of ACSL6 impacts motor function, memory, and age-related neuroinflammation, reflecting the importance of neuronal ACSL6-mediated lipid metabolism across the life span.

CB1 antagonism increases excitatory synaptogenesis in a cortical spheroid model of fetal brain development.

The endocannabinoid system (ECS) plays a complex role in the development of neural circuitry during fetal brain development. The cannabinoid receptor type 1 (CB1) controls synaptic strength at both excitatory and inhibitory synapses and thus contributes to the balance of excitatory and inhibitory signaling. Imbalances in the ratio of excitatory to inhibitory synapses have been implicated in various neuropsychiatric disorders associated with dysregulated central nervous system development including autism spectrum disorder, epilepsy, and schizophrenia. The role of CB1 in human brain development has been difficult to study but advances in induced pluripotent stem cell technology have allowed us to model the fetal brain environment. Cortical spheroids resemble the cortex of the dorsal telencephalon during mid-fetal gestation and possess functional synapses, spontaneous activity, an astrocyte population, and pseudo-laminar organization. We first characterized the ECS using STORM microscopy and observed synaptic localization of components similar to that which is observed in the fetal brain. Next, using the CB1-selective antagonist SR141716A, we observed an increase in excitatory, and to a lesser extent, inhibitory synaptogenesis as measured by confocal image analysis. Further, CB1 antagonism increased the variability of spontaneous activity within developing neural networks, as measured by microelectrode array. Overall, we have established that cortical spheroids express ECS components and are thus a useful model for exploring endocannabinoid mediation of childhood neuropsychiatric disease.

Hyaluronan regulates synapse formation and function in developing neural networks.

Neurodevelopmental disorders present with synaptic alterations that disrupt the balance between excitatory and inhibitory signaling. For example, hyperexcitability of cortical neurons is associated with both epilepsy and autism spectrum disorders. However, the mechanisms that initially establish the balance between excitatory and inhibitory signaling in brain development are not well understood. Here, we sought to determine how the extracellular matrix directs synapse formation and regulates synaptic function in a model of human cortical brain development. The extracellular matrix, making up twenty percent of brain volume, is largely comprised of hyaluronan. Hyaluronan acts as both a scaffold of the extracellular matrix and a space-filling molecule. Hyaluronan is present from the onset of brain development, beginning with neural crest cell migration. Through acute perturbation of hyaluronan levels during synaptogenesis, we sought to determine how hyaluronan impacts the ratio of excitatory to inhibitory synapse formation and the resulting neural activity. We used 3-D cortical spheroids derived from human induced pluripotent stem cells to replicate this neurodevelopmental window. Our results demonstrate that hyaluronan preferentially surrounds nascent excitatory synapses. Removal of hyaluronan increases the expression of excitatory synapse markers and results in a corresponding increase in the formation of excitatory synapses, while also decreasing inhibitory synapse formation. This increased excitatory synapse formation elevates network activity, as demonstrated by microelectrode array analysis. In contrast, the addition of purified hyaluronan suppresses excitatory synapse formation. These results establish that the hyaluronan extracellular matrix surrounds developing excitatory synapses, where it critically regulates synapse formation and the resulting balance between excitatory to inhibitory signaling.

Cytoskeletal regulation of synaptogenesis in a model of human fetal brain development.

Excitatory synapse formation begins in mid-fetal gestation. However, due to our inability to image fetal synaptogenesis, the initial formation of synapses remains understudied. The recent development of human fetal brain spheroids provides access to this critical period of synapse formation. Using human neurons and brain spheroids, we address how altered actin regulation impacts the formation of excitatory synapses during fetal brain development. Prior to synapse formation, inhibition of RhoA kinase (ROCK) signaling promotes neurite elongation and branching. In addition to increasing neural complexity, ROCK inhibition increases the length of protrusions along the neurite, ultimately promoting excitatory synapse formation in human cortical brain spheroids. A corresponding increase in Rac1-driven actin polymerization drives this increase in excitatory synaptogenesis. Using STORM super-resolution microscopy, we demonstrate that actomyosin regulators, including the Rac1 regulator, α-PIX, and the RhoA regulator, p115-RhoGEF, localize to nascent excitatory synapses, where they preferentially localize to postsynaptic compartments. These results demonstrate that coordinated RhoGTPase activities underlie the initial formation of excitatory synapses and identify critical cytoskeletal regulators of early synaptogenic events.

Human-Derived Brain Models: Windows into Neuropsychiatric Disorders and Drug Therapies.

Human-derived neurons and brain organoids have revolutionized our ability to model brain development in a dish. In this review, we discuss the potential for human brain models to advance drug discovery for complex neuropsychiatric disorders. First, we address the advantages of human brain models to screen for new drugs capable of altering CNS activity. Next, we propose an experimental pipeline for using human-derived neurons and brain organoids to rapidly assess drug impact on key events in brain development, including neurite extension, synapse formation, and neural activity. The experimental pipeline begins with automated high content imaging for analysis of neurites, synapses, and neuronal viability. Following morphological examination, multi-well microelectrode array technology examines neural activity in response to drug treatment. These techniques can be combined with high throughput sequencing and mass spectrometry to assess associated transcriptional and proteomic changes. These combined technologies provide a foundation for neuropsychiatric drug discovery and future clinical assessment of patient-specific drug responses.

The effect of rho kinase inhibition on morphological and electrophysiological maturity in iPSC-derived neurons.

Induced pluripotent stem cell (iPSC)-derived neurons permit the study of neurogenesis and neurological disease in a human setting. However, the electrophysiological properties of iPSC-derived neurons are consistent with those observed in immature cortical neurons, including a high membrane resistance depolarized resting membrane potential and immature firing properties, limiting their use in modeling neuronal activity in adult cells. Based on the proven association between inhibiting rho kinase (ROCK) and increased neurite complexity, we seek to determine if short-term ROCK inhibition during the first 1-2 weeks of differentiation would increase morphological complexity and electrophysiological maturity after several weeks of differentiation. While inhibiting ROCK resulted in increased neurite formation after 24 h, this effect did not persist at 3 and 6 weeks of age. Additionally, there was no effect of ROCK inhibition on electrophysiological properties at 2-3, 6, or 12 weeks of age, despite an increase in evoked and spontaneous firing and a more hyperpolarized resting membrane potential over time. These results indicate that while there is a clear effect of time on electrophysiological maturity, ROCK inhibition did not accelerate maturity.

Stem cell models of human synapse development and degeneration.

Many brain disorders exhibit altered synapse formation in development or synapse loss with age. To understand the complexities of human synapse development and degeneration, scientists now engineer neurons and brain organoids from human-induced pluripotent stem cells (hIPSC). These hIPSC-derived brain models develop both excitatory and inhibitory synapses and functional synaptic activity. In this review, we address the ability of hIPSC-derived brain models to recapitulate synapse development and insights gained into the molecular mechanisms underlying synaptic alterations in neuronal disorders. We also discuss the potential for more accurate human brain models to advance our understanding of synapse development, degeneration, and therapeutic responses.

Dynamic cytoplasmic projections connect mammalian spermatogonia in vivo.

Throughout the male reproductive lifespan, spermatogonial stem cells (SSCs) produce committed progenitors that proliferate and then remain physically connected in growing clones via short cylindrical intercellular bridges (ICBs). These ICBs, which enlarge in meiotic spermatocytes, have been demonstrated to provide a conduit for postmeiotic haploid spermatids to share sex chromosome-derived gene products. In addition to ICBs, spermatogonia exhibit multiple thin cytoplasmic projections. Here, we have explored the nature of these projections in mice and find that they are dynamic, span considerable distances from their cell body (≥25 µm), either terminate or physically connect multiple adjacent spermatogonia, and allow for sharing of macromolecules. Our results extend the current model that subsets of spermatogonia exist as isolated cells or clones, and support a model in which spermatogonia of similar developmental fates are functionally connected through a shared dynamic cytoplasm mediated by thin cytoplasmic projections.

RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development.

Small RhoGTPases regulate changes in post-synaptic spine morphology and density that support learning and memory. They are also major targets of synaptic disorders, including Autism. Here we sought to determine whether upstream RhoGTPase regulators, including GEFs, GAPs, and GDIs, sculpt specific stages of synaptic development. The majority of examined molecules uniquely regulate either early spine precursor formation or later maturation. Specifically, an activator of actin polymerization, the Rac1 GEF ß-PIX, drives spine precursor formation, whereas both FRABIN, a Cdc42 GEF, and OLIGOPHRENIN-1, a RhoA GAP, regulate spine precursor elongation. However, in later development, a novel Rac1 GAP, ARHGAP23, and RhoGDIs inactivate actomyosin dynamics to stabilize mature synapses. Our observations demonstrate that specific combinations of RhoGTPase regulatory proteins temporally balance RhoGTPase activity during post-synaptic spine development.

Breaking down to build up: Neuroligin's C-terminal domain strengthens the synapse.

The mechanisms by which neuroligin adhesion molecules modulate synaptic plasticity remain unclear. In this issue, Liu et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201509023) demonstrate that neuroligin 1 promotes actin assembly associated with synaptic strengthening independent of adhesion, suggesting additional ways for neuroligins to contribute to neuronal development and disease pathology.

Non-muscle myosin II in disease: mechanisms and therapeutic opportunities.

The actin motor protein non-muscle myosin II (NMII) acts as a master regulator of cell morphology, with a role in several essential cellular processes, including cell migration and post-synaptic dendritic spine plasticity in neurons. NMII also generates forces that alter biochemical signaling, by driving changes in interactions between actin-associated proteins that can ultimately regulate gene transcription. In addition to its roles in normal cellular physiology, NMII has recently emerged as a critical regulator of diverse, genetically complex diseases, including neuronal disorders, cancers and vascular disease. In the context of these disorders, NMII regulatory pathways can be directly mutated or indirectly altered by disease-causing mutations. NMII regulatory pathway genes are also increasingly found in disease-associated copy-number variants, particularly in neuronal disorders such as autism and schizophrenia. Furthermore, manipulation of NMII-mediated contractility regulates stem cell pluripotency and differentiation, thus highlighting the key role of NMII-based pharmaceuticals in the clinical success of stem cell therapies. In this Review, we discuss the emerging role of NMII activity and its regulation by kinases and microRNAs in the pathogenesis and prognosis of a diverse range of diseases, including neuronal disorders, cancer and vascular disease. We also address promising clinical applications and limitations of NMII-based inhibitors in the treatment of these diseases and the development of stem-cell-based therapies.

ROCK1 and 2 differentially regulate actomyosin organization to drive cell and synaptic polarity.

RhoGTPases organize the actin cytoskeleton to generate diverse polarities, from front-back polarity in migrating cells to dendritic spine morphology in neurons. For example, RhoA through its effector kinase, RhoA kinase (ROCK), activates myosin II to form actomyosin filament bundles and large adhesions that locally inhibit and thereby polarize Rac1-driven actin polymerization to the protrusions of migratory fibroblasts and the head of dendritic spines. We have found that the two ROCK isoforms, ROCK1 and ROCK2, differentially regulate distinct molecular pathways downstream of RhoA, and their coordinated activities drive polarity in both cell migration and synapse formation. In particular, ROCK1 forms the stable actomyosin filament bundles that initiate front-back and dendritic spine polarity. In contrast, ROCK2 regulates contractile force and Rac1 activity at the leading edge of migratory cells and the spine head of neurons; it also specifically regulates cofilin-mediated actin remodeling that underlies the maturation of adhesions and the postsynaptic density of dendritic spines.

Myosin IIb activity and phosphorylation status determines dendritic spine and post-synaptic density morphology.

Dendritic spines in hippocampal neurons mature from a filopodia-like precursor into a mushroom-shape with an enlarged post-synaptic density (PSD) and serve as the primary post-synaptic location of the excitatory neurotransmission that underlies learning and memory. Using myosin II regulatory mutants, inhibitors, and knockdowns, we show that non-muscle myosin IIB (MIIB) activity determines where spines form and whether they persist as filopodia-like spine precursors or mature into a mushroom-shape. MIIB also determines PSD size, morphology, and placement in the spine. Local inactivation of MIIB leads to the formation of filopodia-like spine protrusions from the dendritic shaft. However, di-phosphorylation of the regulatory light chain on residues Thr18 and Ser19 by Rho kinase is required for spine maturation. Inhibition of MIIB activity or a mono-phosphomimetic mutant of RLC similarly prevented maturation even in the presence of NMDA receptor activation. Expression of an actin cross-linking, non-contractile mutant, MIIB R709C, showed that maturation into a mushroom-shape requires contractile activity. Loss of MIIB also leads to an elongated PSD morphology that is no longer restricted to the spine tip; whereas increased MIIB activity, specifically through RLC-T18, S19 di-phosphorylation, increases PSD area. These observations support a model whereby myosin II inactivation forms filopodia-like protrusions that only mature once NMDA receptor activation increases RLC di-phosphorylation to stimulate MIIB contractility, resulting in mushroom-shaped spines with an enlarged PSD.

Cell migration: PKA and RhoA set the pace.

A new study shows that protein kinase A (PKA) activity establishes a signaling loop that governs protrusion-retraction cycles in migrating cells. PKA activity near the leading edge of protrusions phosphorylates RhoA and inhibits its activity via increased association with RhoGDI.

Myosin IIA/IIB restrict adhesive and protrusive signaling to generate front-back polarity in migrating cells.

Migratory front-back polarity emerges from the cooperative effect of myosin IIA (MIIA) and IIB (MIIB) on adhesive signaling. We demonstrate here that, during polarization, MIIA and MIIB coordinately promote localized actomyosin bundling, which generates large, stable adhesions that do not signal to Rac and thereby form the cell rear. MIIA formed dynamic actomyosin proto-bundles that mark the cell rear during spreading; it also bound to actin filament bundles associated with initial adhesion maturation in protrusions. Subsequent incorporation of MIIB stabilized the adhesions and actomyosin filaments with which it associated and formed a stable, extended rear. These adhesions did not turn over and no longer signal to Rac. Microtubules fine-tuned the polarity by positioning the front opposite the MIIA/MIIB-specified rear. Decreased Rac signaling in the vicinity of the MIIA/MIIB-stabilized proto-bundles and adhesions was accompanied by the loss of Rac guanine nucleotide exchange factor (GEFs), like ßPIX and DOCK180, and by inhibited phosphorylation of key residues on adhesion proteins that recruit and activate Rac GEFs. These observations lead to a model for front-back polarity through local GEF depletion.