A mathematical study of the efficacy of possible negative feedback pathways involved in neuronal polarization.

Neuronal polarization, a process wherein nascent neurons develop a single long axon and multiple short dendrites, can occur within in vitro cell cultures without environmental cues. This is an apparently random process in which one of several short processes, called neurites, grows to become long, while the others remain short. In this study, we propose a minimum model for neurite growth, which involves bistability and random excitations reflecting actin waves. Positive feedback is needed to produce the bistability, while negative feedback is required to ensure that no more than one neurite wins the winner-takes-all contest. By applying the negative feedback to different aspects of the neurite growth process, we demonstrate that targeting the negative feedback to the excitation amplitude results in the most persistent polarization. Also, we demonstrate that there are optimal ranges of values for the neurite count, and for the excitation rate and amplitude that best maintain the polarization. Finally, we show that a previously published model for neuronal polarization based on competition for limited resources shares key features with our best-performing minimal model: bistability and negative feedback targeted to the size of random excitations.

STK25 and MST3 Have Overlapping Roles to Regulate Rho GTPases during Cortical Development.

Precise control of neuronal migration is required for the laminar organization of the neocortex and critical for brain function. We previously reported that the acute disruption of the Stk25 gene (Stk25 conditional knock-out; cKO) during mouse embryogenesis causes anomalous neuronal migration in the neocortex, but paradoxically the Stk25 cKO did not have a cortical phenotype, suggesting some forms of compensation exist. In this study, we report that MST3, another member of the GCKIII subgroup of the Ste20-like kinase family, compensates for loss of Stk25 and vice versa with sex independent manner. MST3 overexpression rescued neuronal migration deficit and abnormal axonogenesis in Stk25 cKO brains. Mechanistically, STK25 leads to Rac1 activation and reduced RhoA levels in the developing brain, both of which are required to fully restore neuronal migration in the Stk25 cKO brain. Abnormal migration phenotypes are also rescued by overexpression of Bacurd1and Cul3, which target RhoA for degradation, and activate Rac1. This study reveals that MST3 upregulation is capable of rescuing acute Stk25 deficiency and resolves details of signaling downstream STK25 required for corticogenesis both common to and distinct from MST3 signaling.SIGNIFICANCE STATEMENT Proper neuronal migration during cortical development is required for normal neuronal function. Here, we show that STK25 and MST3 kinases regulate neuronal migration and polarization in a mutually compensatory manner. Furthermore, STK25 balances Rac1 activity and RhoA level through forming complexes with α-PIX and ß-PIX, GTPase regulatory enzymes, and Cullin3-Bacurd1/Kctd13, a pair of RhoA ubiquitination molecules in a kinase activity-independent manner. Our findings demonstrate the importance of overlapping and unique roles of STK25 and MST3 to regulate Rho GTPase activities in cortical development.

Talin promotes integrin activation accompanied by generation of tension in talin and an increase in osmotic pressure in neurite outgrowth.

Neuronal polarization depends on the interaction of intracellular chemical and mechanical activities in which the cytoplasmic protein, talin, plays a pivotal role during neurite growth. To better understand the mechanism underlying talin function in neuronal polarization, we overexpressed several truncated forms of talin and found that the presence of the rod domain within the overexpressed talin is required for its positive effect on neurite elongation because the neurite number only increased when the talin head region was overexpressed. The tension in the talin rod was recognized using a Förster resonance energy transfer-based tension probe. Nerve growth factor treatment resulted in inward tension of talin elicited by microfilament force and outward osmotic pressure. By contrast, the glial scar-inhibitor aggrecan weakened these forces, suggesting that interactions between inward pull forces in the talin rod and outward osmotic pressure participate in neuronal polarization. Integrin activation is also involved in up-regulation of talin tension and osmotic pressure. Aggrecan stimuli resulted in up-regulation of docking protein 1 (DOK1), leading to the down-regulation of integrin activity and attenuation of the intracellular mechanical force. Our study suggests interactions between the intracellular inward tension in talin and the outward osmotic pressure as the effective channel for promoting neurite outgrowth, which can be up-regulated by integrin activation and down-regulated by DOK1.-Wang, Y., Zhang, X., Tian, J., Shan, J., Hu, Y., Zhai, Y., Guo, J. Talin promotes integrin activation accompanied by generation of tension in talin and an increase in osmotic pressure in neurite outgrowth.

Protein kinase LKB1 regulates polarized dendrite formation of adult hippocampal newborn neurons.

Adult-born granule cells in the dentate gyrus of the rodent hippocampus are important for memory formation and mood regulation, but the cellular mechanism underlying their polarized development, a process critical for their incorporation into functional circuits, remains unknown. We found that deletion of the serine-threonine protein kinase LKB1 or overexpression of dominant-negative LKB1 reduced the polarized initiation of the primary dendrite from the soma and disrupted its oriented growth toward the molecular layer. This abnormality correlated with the dispersion of Golgi apparatus that normally accumulated at the base and within the initial segment of the primary dendrite, and was mimicked by disrupting Golgi organization via altering the expression of Golgi structural proteins GM130 or GRASP65. Thus, besides its known function in axon formation in embryonic pyramidal neurons, LKB1 plays an additional role in regulating polarized dendrite morphogenesis in adult-born granule cells in the hippocampus.

Spatial gene's (Tbata) implication in neurite outgrowth and dendrite patterning in hippocampal neurons.

The unique architecture of neurons requires the establishment and maintenance of polarity, which relies in part on microtubule-based kinesin motor transport to deliver essential cargo into axons and dendrites. In developing neurons, kinesin trafficking is essential for delivering organelles and molecules that are crucial for elongation and guidance of the growing axonal and dendritic termini. In mature neurons, kinesin cargo delivery is essential for neuron dynamic physiological functions which are critical in brain development. In this work, we followed Spatial (Tbata) gene expression during primary hippocampal neuron development and showed that it is highly expressed during dendrite formation. Spatial protein exhibits a somatodendritic distribution and we show that the kinesin motor Kif17, among other dendrite specific kinesins, is crucial for Spatial localization to dendrites of hippocampal neurons. Furthermore, Spatial down regulation in primary hippocampal cells revealed a role for Spatial in maintaining neurons' polarity by ensuring proper neurite outgrowth. This polarity is specified by intrinsic and extracellular signals that allow neurons to determine axon and dendrite fate during development. Neurotrophic factors, such as the Nerve Growth Factor (NGF), are candidate extracellular polarity-regulating cues which are proposed to accelerate neuronal polarization by enhancing dendrite growth. Here, we show that NGF treatment increases Spatial expression in hippocampal neurons. Altogether, these data suggest that Spatial, in response to NGF and through its transport by Kif17, is crucial for neuronal polarization and can be a key regulator of neurite outgrowth.

Integrative multi-omic analysis reveals conserved cell-projection deficits in human Down syndrome brains.

Down syndrome (DS) is the most common genetic cause of cognitive disability. However, it is largely unclear how triplication of a small gene subset may impinge on diverse aspects of DS brain physiopathology. Here, we took a multi-omic approach and simultaneously analyzed by RNA-seq and proteomics the expression signatures of two diverse regions of human postmortem DS brains. We found that the overexpression of triplicated genes triggered global expression dysregulation, differentially affecting transcripts, miRNAs, and proteins involved in both known and novel biological candidate pathways. Among the latter, we observed an alteration in RNA splicing, specifically modulating the expression of genes involved in cytoskeleton and axonal dynamics in DS brains. Accordingly, we found an alteration in axonal polarization in neurons from DS human iPSCs and mice. Thus, our study provides an integrated multilayer expression database capable of identifying new potential targets to aid in designing future clinical interventions for DS.

Emergent structural and functional properties of hippocampal multi-cellular aggregates.

Hippocampal neural networks are distinctly capable of integrating multi-modal sensory inputs to drive memory formation. Neuroscientific investigations using simplified in vitro models have greatly relied on planar (2D) neuronal cultures made from dissociated tissue. While these models have served as simple, cost-effective, and high-throughput tools for examining various morphological and electrophysiological characteristics of hippocampal networks, 2D cultures fail to reconstitute critical elements of the brain microenvironment that may be necessary for the emergence of sophisticated integrative network properties. To address this, we utilized a forced aggregation technique to generate high-density (>100,000 cells/mm3) multi-cellular three-dimensional aggregates using rodent embryonic hippocampal tissue. We contrasted the emergent structural and functional properties of aggregated (3D) and dissociated (2D) cultures over 28 days in vitro (DIV). Hippocampal aggregates displayed robust axonal fasciculation across large distances and significant neuronal polarization, i.e., spatial segregation of dendrites and axons, at earlier time points compared to dissociated cultures. Moreover, we found that astrocytes in aggregate cultures self-organized into non-overlapping quasi-domains and developed highly stellate morphologies resembling astrocyte structures in vivo. We maintained cultures on multi-electrode arrays (MEAs) to assess spontaneous electrophysiological activity for up to 28 DIV. We found that 3D networks of aggregated cultures developed highly synchronized networks and with high burstiness by 28 DIV. We also demonstrated that dual-aggregate networks became active by 7 DIV, in contrast to single-aggregate networks which became active and developed synchronous bursting activity with repeating motifs by 14 DIV. Taken together, our findings demonstrate that the high-density, multi-cellular, 3D microenvironment of hippocampal aggregates supports the recapitulation of emergent biofidelic morphological and functional properties. Our findings suggest that neural aggregates may be used as segregated, modular building blocks for the development of complex, multi-nodal neural network topologies.

The kinesin Kif21b regulates radial migration of cortical projection neurons through a non-canonical function on actin cytoskeleton.

Completion of neuronal migration is critical for brain development. Kif21b is a plus-end-directed kinesin motor protein that promotes intracellular transport and controls microtubule dynamics in neurons. Here we report a physiological function of Kif21b during radial migration of projection neurons in the mouse developing cortex. In vivo analysis in mouse and live imaging on cultured slices demonstrate that Kif21b regulates the radial glia-guided locomotion of newborn neurons independently of its motility on microtubules. We show that Kif21b directly binds and regulates the actin cytoskeleton both in vitro and in vivo in migratory neurons. We establish that Kif21b-mediated regulation of actin cytoskeleton dynamics influences branching and nucleokinesis during neuronal locomotion. Altogether, our results reveal atypical roles of Kif21b on the actin cytoskeleton during migration of cortical projection neurons.

Protective Effects of 4-Trifluoromethyl-(E)-cinnamoyl]-L-4-F-phenylalanine Acid against Chronic Cerebral Hypoperfusion Injury through Promoting Brain-Derived Neurotrophic Factor-Mediated Neurogenesis.

Vascular dementia (VaD), one of the major consequences after stroke, is the second reason for the cognitive decline in aged people. Chronic cerebral hypoperfusion (CCH) is considered as the main cause for cognitive impairment in VaD patients. In our previous study, a synthetic compound, 4-trifluoromethyl-(E)-cinnamoyl]-L-4-F-phenylalanine acid (AE-18), has been proven to decrease infarct volume and to recover the insufficient blood supply after ischemia-reperfusion in rats, which was reminded that AE-18 may possess the ameliorative effect in CCH. In this study, the bilateral common carotid artery occlusion was performed to establish the CCH model in rats to evaluate the effect and mechanisms of AE-18 in CCH. Results showed that AE-18 (5 and 10 mg/kg, i.g.) could recover the learning and memory and increase the number of neurons in the hippocampus, which may be attributed to its neurogenesis effects and its recovery of cerebral blood flow in CCH rats. In addition, the in vitro studies showed that AE-18 promoted neuronal proliferation, induced differentiation of Neuro-2a cells into a neuron-like morphology, and accelerated the establishment of axon-dendrite polarization of primary hippocampal neurons through upregulating brain-derived neurotrophic factor via the PI3K/Akt/CREB pathway. In conclusion, AE-18 is a promising candidate for the treatment of cognitive decline after CCH injury by restoring blood supply to the brain and promoting neurogenesis in the hippocampus.

Microglia modulate proliferation, neurite generation and differentiation of human neural progenitor cells.

Microglia, the primary immune cells of the brain, significantly influence the fate of neurons after neural damage. Depending on the local environment, they exhibit a wide range of phenotypes, including patrolling (naïve), proinflammatory, and anti-inflammatory characteristics, which greatly affects neurotoxicity. Despite the fact that neural progenitor cells (NPCs) and hippocampal neurons represent cell populations, which play pivotal role in neural regeneration, interaction between microglia and these cell types is poorly studied. In the present work, we investigated how microglial cells affect the proliferation and neurite outgrowth of human stem cell-derived NPCs, and how microglia stimulation with proinflammatory or anti-inflammatory agents modulates this interaction. We found that naïve microglia slightly diminish NPC proliferation and have no effect on neurite outgrowth. In contrast, proinflammatory stimulated microglia promote both proliferation and neurite generation, whereas microglia stimulated with anti-inflammatory cytokines augment neurite outgrowth leaving NPC proliferation unaffected. We also studied how microglia influence neurite development and differentiation of hippocampal dentate gyrus granule cells differentiated from NPCs. We found that proinflammatory stimulated microglia inhibit axonal development but facilitate dendrite generation in these differentiating neurons. Our results elucidate a fine-tuned modulatory effect of microglial cells on cell types crucial for neural regeneration, opening perspectives for novel regenerative therapeutic interventions.

Axon Growth of CNS Neurons in Three Dimensions Is Amoeboid and Independent of Adhesions.

During development of the central nervous system (CNS), neurons polarize and rapidly extend their axons to assemble neuronal circuits. The growth cone leads the axon to its target and drives axon growth. Here, we explored the mechanisms underlying axon growth in three dimensions. Live in situ imaging and super-resolution microscopy combined with pharmacological and molecular manipulations as well as biophysical force measurements revealed that growth cones extend CNS axons independent of pulling forces on their substrates and without the need for adhesions in three-dimensional (3D) environments. In 3D, microtubules grow unrestrained from the actomyosin cytoskeleton into the growth cone leading edge to enable rapid axon extension. Axons extend and polarize even in adhesion-inert matrices. Thus, CNS neurons use amoeboid mechanisms to drive axon growth. Together with our understanding that adult CNS axons regenerate by reactivating developmental processes, our findings illuminate how cytoskeletal manipulations enable axon regeneration in the adult CNS.

Conventional and Non-Conventional Roles of Non-Muscle Myosin II-Actin in Neuronal Development and Degeneration.

Myosins are motor proteins that use chemical energy to produce mechanical forces driving actin cytoskeletal dynamics. In the brain, the conventional non-muscle myosin II (NMII) regulates actin filament cytoskeletal assembly and contractile forces during structural remodeling of axons and dendrites, contributing to morphology, polarization, and migration of neurons during brain development. NMII isoforms also participate in neurotransmission and synaptic plasticity by driving actin cytoskeletal dynamics during synaptic vesicle release and retrieval, and formation, maturation, and remodeling of dendritic spines. NMIIs are expressed differentially in cerebral non-neuronal cells, such as microglia, astrocytes, and endothelial cells, wherein they play key functions in inflammation, myelination, and repair. Besides major efforts to understand the physiological functions and regulatory mechanisms of NMIIs in the nervous system, their contributions to brain pathologies are still largely unclear. Nonetheless, genetic mutations or deregulation of NMII and its regulatory effectors are linked to autism, schizophrenia, intellectual disability, and neurodegeneration, indicating non-conventional roles of NMIIs in cellular mechanisms underlying neurodevelopmental and neurodegenerative disorders. Here, we summarize the emerging biological roles of NMIIs in the brain, and discuss how actomyosin signaling contributes to dysfunction of neurons and glial cells in the context of neurological disorders. This knowledge is relevant for a deep understanding of NMIIs on the pathogenesis and therapeutics of neuropsychiatric and neurodegenerative diseases.

The Motor KIF5C Links the Requirements of Stable Microtubules and IGF-1 Receptor Membrane Insertion for Neuronal Polarization.

Three early signals of asymmetry have been described to occur in a single neurite of neurons at stage 2 of differentiation (before polarization) and shown to be essential for neuronal polarization: (i) accumulation of stable microtubules, (ii) enrichment of the plasma membrane with activatable IGF-1r, and (iii) polarized transport of the microtubular motor KIF5C. Here, we studied the possible relationship between these three phenomena. Our results show that the activatable (membrane-inserted) IGF-1r and stable microtubules accumulate in the same neurite of cells at stage 2. The polarized insertion of IGF-1r depends on microtubule dynamics as shown using drugs which modify microtubule stability. Silencing of KIF5C expression prevents the polarized insertion of IGF-1r into the neuronal plasmalemma and neuronal polarization. Syntaxin 6 and VAMP4, necessary for the polarized insertion of the IGF-1r, are associated to vesicles carried by the microtubular motor KIF5C and is transported preferentially to the neurite where KIF5C accumulates. We conclude that the enrichment of stable microtubules in the future axon enhances KIF5C-mediated vesicular transport of syntaxin 6 and VAMP4, which in turn mediates the polarized insertion of IGF-1r in the plasmalemma, a key step for neuronal polarization. We herewith establish a mechanistic link between three early polarity events necessary for the establishment of neuronal polarity.

Neurons show the path: tip-to-nucleus communication in filamentous fungal development and pathogenesis.

Multiple fungal species penetrate substrates and accomplish host invasion through the fast, permanent and unidirectional extension of filamentous cells known as hyphae. Polar growth of hyphae results, however, in a significant increase in the distance between the polarity site, which also receives the earliest information about ambient conditions, and nuclei, where adaptive responses are executed. Recent studies demonstrate that these long distances are overcome by signal transduction pathways which convey sensory information from the polarity site to nuclei, controlling development and pathogenesis. The present review compares the striking connections of the mechanisms for long-distance communication in hyphae with those from neurons, and discusses the importance of their study in order to understand invasion and dissemination processes of filamentous fungi, and design strategies for developmental control in the future.

Force Generation by Molecular-Motor-Powered Microtubule Bundles; Implications for Neuronal Polarization and Growth.

The heavily cross-linked microtubule (MT) bundles found in neuronal processes play a central role in the initiation, growth and maturation of axons and dendrites; however, a quantitative understanding of their mechanical function is still lacking. We here developed computer simulations to investigate the dynamics of force generation in 1D bundles of MTs that are cross-linked and powered by molecular motors. The motion of filaments and the forces they exert are investigated as a function of the motor type (unipolar or bipolar), MT density and length, applied load, and motor connectivity. We demonstrate that only unipolar motors (e.g., kinesin-1) can provide the driving force for bundle expansion, while bipolar motors (e.g., kinesin-5) oppose it. The force generation capacity of the bundles is shown to depend sharply on the fraction of unipolar motors due to a percolation transition that must occur in the bundle. Scaling laws between bundle length, force, MT length and motor fraction are presented. In addition, we investigate the dynamics of growth in the presence of a constant influx of MTs. Beyond a short equilibration period, the bundles grow linearly in time. In this growth regime, the bundle extends as one mass forward with most filaments sliding with the growth velocity. The growth velocity is shown to be dictated by the inward flux of MTs, to inversely scale with the load and to be independent of the free velocity of the motors. These findings provide important molecular-level insights into the mechanical function of the MT cytoskeleton in normal axon growth and regeneration after injury.

Second messenger signaling for neuronal polarization: cell mechanics-dependent pattern formation.

Neuronal polarization is a critical step in the neuronal morphogenesis. Despite the identification of several evolutionarily conserved factors for neural polarization, the exact mechanisms by which cells initiate and maintain polarity remain to be characterized. Here, we review the recent progress on the roles of second messengers, specifically the cyclic nucleotides and membrane-associated phospholipids, in the initiation, propagation, and integration of polarization signals, and propose an inhibitor-free model for neural polarization. The characteristic features of neuron polarization include the formation of single axon and multiple dendrites. These features involve chemical and mechanical mechanisms such as reaction-diffusion and tug-of-war, by which second messengers can act in concert to initiate and stabilize the cellular asymmetry. Nevertheless, biochemical factors eliciting the long-range inhibition remain ambiguous. Thus, we provide a simple, inhibitor-free model that can incorporate known cytochemical and cytomechanical factors, and produce features of neuronal polarization in environments provided with minimized extracellular regulators.

Selected SNARE proteins are essential for the polarized membrane insertion of igf-1 receptor and the regulation of initial axonal outgrowth in neurons.

The establishment of polarity necessitates initial axonal outgrowth and, therefore, the addition of new membrane to the axon's plasmalemma. Axolemmal expansion occurs by exocytosis of plasmalemmal precursor vesicles (PPVs) primarily at the neuronal growth cone. Little is known about the SNAREs family proteins involved in the regulation of PPV fusion with the neuronal plasmalemma at early stages of differentiation. We show here that five SNARE proteins (VAMP2, VAMP4, VAMP7, Syntaxin6 and SNAP23) were expressed by hippocampal pyramidal neurons before polarization. Expression silencing of three of these proteins (VAMP4, Syntaxin6 and SNAP23) repressed axonal outgrowth and the establishment of neuronal polarity, by inhibiting IGF-1 receptor exocytotic polarized insertion, necessary for neuronal polarization. In addition, stimulation with IGF-1 triggered the association of VAMP4, Syntaxin6 and SNAP23 to vesicular structures carrying the IGF-1 receptor and overexpression of a negative dominant form of Syntaxin6 significantly inhibited exocytosis of IGF-1 receptor containing vesicles at the neuronal growth cone. Taken together, our results indicated that VAMP4, Syntaxin6 and SNAP23 functions are essential for regulation of PPV exocytosis and the polarized insertion of IGF-1 receptor and, therefore, required for initial axonal elongation and the establishment of neuronal polarity.

A natural diarylheptanoid promotes neuronal differentiation via activating ERK and PI3K-Akt dependent pathways.

Neuronal differentiation is a critical developmental process that determines accurate synaptic connection and circuit wiring. A wide variety of naturally occurring compounds have been shown as promising drug leads for the generation and differentiation of neurons. Here we report that a diarylheptanoid from the plant Alpinia officinarum, 7-(4-hydroxyphenyl)-1-phenyl-4E-hepten-3-one (Cpd 1), exhibited potent activities in neuronal differentiation and neurite outgrowth. Cpd 1 induced differentiation of neuroblastoma Neuro-2a cells into a neuron-like morphology, and accelerated the establishment of axon-dendrite polarization of cultured hippocampal neurons. Moreover, Cpd 1 promoted neurite extension in both Neuro-2a cells and neurons. We showed that the effects of Cpd 1 on neuronal differentiation and neurite growth were specifically dependent on the activation of extracellular signal-regulated kinases (ERKs) and phosphoinositide 3-kinase (PI3K)-Akt signaling pathways. Importantly, intraperitoneal administration of Cpd 1 promoted the differentiation of new-born progenitor cells into mature neurons in the adult hippocampal dentate gyrus. Collectively, this study identifies a naturally occurring diarylheptanoid with beneficial effects on neuronal differentiation and neurite outgrowth in vitro and in vivo.

CRMP2: functional roles in neural development and therapeutic potential in neurological diseases.

Cytoskeletal restructuring is essential for nearly all cellular processes in the developing brain. After cell fate determination, newborn cortical neurons must migrate to their final positions while establishing proper axon-dendrite polarity. Significant progress has recently been made towards understanding the cellular and molecular mechanisms underlying neuronal polarization in vivo. Collapsin response mediator protein 2 (CRMP2) has long been identified as a microtubule-binding protein that regulates neuronal polarity in vitro. Recent studies provide new insights into the roles of CRMP2 in neuronal migration and subsequent neuronal differentiation. Both the expression and activity of CRMP2 are tightly regulated during cortex development. CRMP2 is suggested to be important in the multipolar-bipolar transition in radial migration. The increasing number of known interaction partners indicates that CRMP2 has functions beyond cytoskeletal regulation, including axonal transport, vesicle trafficking, and neurotransmitter release. This review discusses the current knowledge about CRMP2 in the context of neuronal development and highlights a recent emerging theme regarding its potential therapeutic applications.