The GTPase Rab21 is required for neuronal development and migration in the cerebral cortex.

Development of the mammalian neocortex requires proper inside-out migration of developing cortical neurons from the germinal ventricular zone toward the cortical plate. The mechanics of this migration requires precise coordination of different cellular phenomena including cytoskeleton dynamics, membrane trafficking, and cell adhesion. The small GTPases play a central role in all these events. The small GTPase Rab21 regulates migration and neurite growth in developing neurons. Moreover, regulators and effectors of Rab21 have been implicated in brain pathologies with cortical malformations, suggesting a key function for the Rab21 signaling pathway in cortical development. Mechanistically, it has been posited that Rab21 influences cell migration by controlling the trafficking of endocytic vesicles containing adhesion molecules. However, direct evidence of the participation of Rab21 or its mechanism of action in the regulation of cortical migration is still incomplete. In this study, we demonstrate that Rab21 plays a critical role in the differentiation and migration of pyramidal neurons by regulating the levels of the amyloid precursor protein on the neuronal cell surface. Rab21 loss of function increased the levels of membrane-exposed APP, resulting in impaired cortical neuronal differentiation and migration. These findings further our understanding of the processes governing the development of the cerebral cortex and shed light onto the molecular mechanisms behind cortical development disorders derived from the malfunctioning of Rab21 signaling effectors.

BARS Influences Neuronal Development by Regulation of Post-Golgi Trafficking.

Neurons are highly polarized cells requiring precise regulation of trafficking and targeting of membrane proteins to generate and maintain different and specialized compartments, such as axons and dendrites. Disruption of the Golgi apparatus (GA) secretory pathway in developing neurons alters axon/dendritic formation. Therefore, detailed knowledge of the mechanisms underlying vesicles exiting from the GA is crucial for understanding neuronal polarity. In this study, we analyzed the role of Brefeldin A-Ribosylated Substrate (CtBP1-S/BARS), a member of the C-terminal-binding protein family, in the regulation of neuronal morphological polarization and the exit of membrane proteins from the Trans Golgi Network. Here, we show that BARS is expressed during neuronal development in vitro and that RNAi suppression of BARS inhibits axonal and dendritic elongation in hippocampal neuronal cultures as well as largely perturbed neuronal migration and multipolar-to-bipolar transition during cortical development in situ. In addition, using plasma membrane (PM) proteins fused to GFP and engineered with reversible aggregation domains, we observed that expression of fission dominant-negative BARS delays the exit of dendritic and axonal membrane protein-containing carriers from the GA. Taken together, these data provide the first set of evidence suggesting a role for BARS in neuronal development by regulating post-Golgi membrane trafficking.

Stress-induced vulnerability to develop cocaine addiction depends on cofilin modulation.

Actin dynamics in dendritic spines can be associated with the neurobiological mechanisms supporting the comorbidity between stress exposure and cocaine increase rewards. The actin cytoskeleton remodeling in the nucleus accumbens (NA) has been implicated in the expression of stress-induced cross-sensitization with cocaine. The present study evaluates the involvement of cofilin, a direct regulator of actin dynamics, in the impact of stress on vulnerability to cocaine addiction. We assess whether the neurobiological mechanisms that modulate repeated-cocaine administration also occur in a chronic restraint stress-induced cocaine self-administration model. We also determine if chronic stress induces alterations in dendritic spines through dysregulation of cofilin activity in the NA core. Here, we show that the inhibition of cofilin expression in the NA core using viral short-hairpin RNA is sufficient to prevent the cocaine sensitization induced by chronic stress. The reduced cofilin levels also impede a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor surface expression enhancement and promote the reduction of head diameter in animals pre-exposed to stress after a cocaine challenge in the NA core. Moreover, downregulation of cofilin expression prevents facilitation of the acquisition of cocaine self-administration (SA) in male rats pre-exposed to chronic stress without modifying performance in sucrose SA. These findings reveal a novel, crucial role for cofilin in the neurobiological mechanisms underpinning the comorbidity between stress exposure and addiction-related disorders.

Molecular Modulation by Lentivirus-Delivered Specific shRNAs in Endoplasmic Reticulum Stressed Neurons.

The accumulation of unfolded proteins within the endoplasmic reticulum (ER), caused by any stress condition, triggers the unfolded protein response (UPR) through the activation of specialized sensors. UPR attempts first to restore homeostasis; but if damage persists the signaling induces apoptosis. There is increasing evidence that sustained and unresolved ER stress contributes to many pathological conditions including neurodegenerative diseases. Because the UPR controls cell fate by switching between cytoprotective and apoptotic processes, it is essential to understand the events defining this transition, as well as the elements involved in its modulation. Recently, we demonstrated that abnormal GM2 ganglioside accumulation causes depletion of ER Ca2+ content, which in turn activates PERK (PKR-like-ER kinase), one of the UPR sensors. Furthermore, PERK signaling participates in the neurite atrophy and apoptosis induced by GM2 accumulation. In this respect, we have established an experimental system that allows us to molecularly modulate the expression of downstream PERK components and thus change vulnerability of neurons to undergo neuritic atrophy. We performed knockdown of calcineurin (cytoprotective) and CHOP (pro-apoptotic) expression in rat cortical neuronal cultures. Cells were infected with lentivirus-delivered specific shRNA and then treated with GM2 at different times, fixed and immunostained with anti-MAP2 (microtube-associated protein 2) antibody. Later, cell images were recorded using a fluorescence microscope and total neurite outgrowth was evaluated by using the public domain image processing software ImageJ. The inhibition of expression of those PERK signaling components clearly made it possible to either accelerate or delay the neuritic atrophy induced by ER stress. This approach might be used in cell system models of ER stress to evaluate the vulnerability of neurons to neurite atrophy.

The neuronal ceroid lipofuscinosis-related protein CLN8 regulates endo-lysosomal dynamics and dendritic morphology.

BACKGROUND INFORMATION: The endo-lysosomal system (ELS) comprises a set of membranous organelles responsible for transporting intracellular and extracellular components within cells. Defects in lysosomal proteins usually affect a large variety of processes and underlie many diseases, most of them with a strong neuronal impact. Mutations in the endoplasmic reticulum-resident CLN8 protein cause CLN8 disease. This condition is one of the 14 known neuronal ceroid lipofuscinoses (NCLs), a group of inherited diseases characterised by accumulation of lipofuscin-like pigments within lysosomes. Besides mediating the transport of soluble lysosomal proteins, recent research suggested a role for CLN8 in the transport of vesicles and lipids, and autophagy. However, the consequences of CLN8 deficiency on ELS structure and activity, as well as the potential impact on neuronal development, remain poorly characterised. Therefore, we performed CLN8 knockdown in neuronal and non-neuronal cell models to analyse structural, dynamic and functional changes in the ELS and to assess the impact of CLN8 deficiency on axodendritic development. RESULTS: CLN8 knockdown increased the size of the Golgi apparatus, the number of mobile vesicles and the speed of endo-lysosomes. Using the fluorescent fusion protein mApple-LAMP1-pHluorin, we detected significant lysosomal alkalisation in CLN8-deficient cells. In turn, experiments in primary rat hippocampal neurons showed that CLN8 deficiency decreased the complexity and size of the somatodendritic compartment. CONCLUSIONS: Our results suggest the participation of CLN8 in vesicular distribution, lysosomal pH and normal development of the dendritic tree. We speculate that the defects triggered by CLN8 deficiency on ELS structure and dynamics underlie morphological alterations in neurons, which ultimately lead to the characteristic neurodegeneration observed in this NCL. SIGNIFICANCE: This is, to our knowledge, the first characterisation of the effects of CLN8 dysfunction on the structure and dynamics of the ELS. Moreover, our findings suggest a novel role for CLN8 in somatodendritic development, which may account at least in part for the neuropathological manifestations associated with CLN8 disease.

Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using fluorescence microscopy.

Fluorescent nanoscopy approaches have been used to characterize the periodic organization of actin, spectrin and associated proteins in neuronal axons and dendrites. This membrane-associated periodic skeleton (MPS) is conserved across animals, suggesting it is a fundamental component of neuronal extensions. The nanoscale architecture of the arrangement (190 nm) is below the resolution limit of conventional fluorescent microscopy. Fluorescent nanoscopy, on the other hand, requires costly equipment and special analysis routines, which remain inaccessible to most research groups. This report aims to resolve this issue by using protein-retention expansion microscopy (pro-ExM) to reveal the MPS of axons. ExM uses reagents and equipment that are readily accessible in most neurobiology laboratories. We first explore means to accurately estimate the expansion factors of protein structures within cells. We then describe the protocol that produces an expanded specimen that can be examined with any fluorescent microscopy allowing quantitative nanoscale characterization of the MPS. We validate ExM results by direct comparison to stimulated emission depletion (STED) nanoscopy. We conclude that ExM facilitates three-dimensional, multicolor and quantitative characterization of the MPS using accessible reagents and conventional fluorescent microscopes.

Neurotoxicity of the pesticide rotenone on neuronal polarization: a mechanistic approach.

Neurons are the most extensive and polarized cells that display a unique single long axon and multiple dendrites, which are compartments exhibiting structural and functional differences. Polarity occurs early in neuronal development and it is maintained by complex subcellular mechanisms throughout cell life. A well-defined and controlled spatio-temporal program of cellular and molecular events strictly regulates the formation of the axon and dendrites from a non-polarized cell. This event is critical for an adequate neuronal wiring and therefore for the normal functioning of the nervous system. Neuronal polarity is very sensitive to the harmful effects of different factors present in the environment. In this regard, rotenone is a crystalline, colorless and odorless isoflavone used as insecticide, piscicide and broad spectrum pesticide commonly used earlier in agriculture. In the present review we will summarize the toxicity mechanism caused by this pesticide in different neuronal cell types, focusing on a particular biological mechanism whereby rotenone could impair neuronal polarization in cultured hippocampal neurons. Recent advances suggest that the inhibition of axonogenesis produced by rotenone could be related with its effect on microtubule dynamics, the actin cytoskeleton and their regulatory pathways, particularly affecting the small RhoGTPase RhoA. Unveiling the mechanism by which rotenone produces neurotoxicity will be instrumental to understand the cellular mechanisms involved in neurodegenerative diseases influenced by this environmental pollutant, which may lead to research focused on the design of new therapeutic strategies.

A key function for microtubule-associated-protein 6 in activity-dependent stabilisation of actin filaments in dendritic spines.

Emerging evidence indicates that microtubule-associated proteins (MAPs) are implicated in synaptic function; in particular, mice deficient for MAP6 exhibit striking deficits in plasticity and cognition. How MAP6 connects to plasticity mechanisms is unclear. Here, we address the possible role of this protein in dendritic spines. We find that in MAP6-deficient cortical and hippocampal neurons, maintenance of mature spines is impaired, and can be restored by expressing a stretch of the MAP6 sequence called Mc modules. Mc modules directly bind actin filaments and mediate activity-dependent stabilisation of F-actin in dendritic spines, a key event of synaptic plasticity. In vitro, Mc modules enhance actin filament nucleation and promote the formation of stable, highly ordered filament bundles. Activity-induced phosphorylation of MAP6 likely controls its transfer to the spine cytoskeleton. These results provide a molecular explanation for the role of MAP6 in cognition, enlightening the connection between cytoskeletal dysfunction, synaptic impairment and neuropsychiatric illnesses.

Rotenone inhibits axonogenesis via an Lfc/RhoA/ROCK pathway in cultured hippocampal neurons.

Rotenone, a broad-spectrum insecticide, piscicide and pesticide, produces a complete and selective suppression of axonogenesis in cultured hippocampal neurons. This effect is associated with an inhibition of actin dynamics through activation of Ras homology member A (RhoA) activity. However, the upstream signaling mechanisms involved in rotenone-induced RhoA activation were unknown. We hypothesized that rotenone might inhibit axon growth by the activation of RhoA/ROCK pathway because of the changes in microtubule (MT) dynamics and the concomitant release of Lfc, a MT-associated Guanine Nucleotide Exchange Factor (GEF) for RhoA. In this study, we demonstrate that rotenone decreases MT stability in morphologically unpolarized neurons. Taxol (3 nM), a drug that stabilizes MT, attenuates the inhibitory effect of rotenone (0.1 µM) on axon formation. Radiometric Forster Resonance Energy Transfer, revealed that this effect is associated with inhibition of rotenone-induced RhoA and ROCK activation. Interestingly, silencing of Lfc, but not of the RhoA GEF ArhGEF1, prevents the inhibitory effect of rotenone on axon formation. Our results suggest that rotenone-induced MT de-stabilization releases Lfc from MT thereby promoting RhoA and ROCK activities and the consequent inhibition of axon growth. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Author Correction: Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration.

Axonal degeneration occurs in the developing nervous system for the appropriate establishment of mature circuits, and is also a hallmark of diverse neurodegenerative diseases. Despite recent interest in the field, little is known about the changes (and possible role) of the cytoskeleton during axonal degeneration. We studied the actin cytoskeleton in an in vitro model of developmental pruning induced by trophic factor withdrawal (TFW). We found that F-actin decrease and growth cone collapse (GCC) occur early after TFW; however, treatments that prevent axonal fragmentation failed to prevent GCC, suggesting independent pathways. Using super-resolution (STED) microscopy we found that the axonal actin/spectrin membrane-associated periodic skeleton (MPS) abundance and organization drop shortly after deprivation, remaining low until fragmentation. Fragmented axons lack MPS (while maintaining microtubules) and acute pharmacological treatments that stabilize actin filaments prevent MPS loss and protect from axonal fragmentation, suggesting that MPS destruction is required for axon fragmentation to proceed.

Regulation of plasma membrane expansion during axon formation.

Here, will review current evidence regarding the signaling pathways and mechanisms underlying membrane addition at sites of active growth during axon formation. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 170-180, 2018.

Prion protein inhibits fast axonal transport through a mechanism involving casein kinase 2.

Prion diseases include a number of progressive neuropathies involving conformational changes in cellular prion protein (PrPc) that may be fatal sporadic, familial or infectious. Pathological evidence indicated that neurons affected in prion diseases follow a dying-back pattern of degeneration. However, specific cellular processes affected by PrPc that explain such a pattern have not yet been identified. Results from cell biological and pharmacological experiments in isolated squid axoplasm and primary cultured neurons reveal inhibition of fast axonal transport (FAT) as a novel toxic effect elicited by PrPc. Pharmacological, biochemical and cell biological experiments further indicate this toxic effect involves casein kinase 2 (CK2) activation, providing a molecular basis for the toxic effect of PrPc on FAT. CK2 was found to phosphorylate and inhibit light chain subunits of the major motor protein conventional kinesin. Collectively, these findings suggest CK2 as a novel therapeutic target to prevent the gradual loss of neuronal connectivity that characterizes prion diseases.

Imaging Golgi Outposts in Fixed and Living Neurons.

Here we describe the use of confocal microscopy in combination with antibodies specific to Golgi proteins to visualize dendritic Golgi outposts (GOPs) in cultured hippocampal pyramidal neurons. We also describe the use of spinning disk confocal microscopy, in combination with ectopically expressed glycosyltransferases fused to GFP variants, to visualize GOPs in living neurons.

A RhoA Signaling Pathway Regulates Dendritic Golgi Outpost Formation.

The neuronal Golgi apparatus (GA) localizes to the perinuclear region and dendrites as tubulo-vesicular structures designated Golgi outposts (GOPs). Current evidence suggests that GOPs shape dendrite morphology and serve as platforms for the local delivery of synaptic receptors. However, the mechanisms underlying GOP formation remain a mystery. Using live-cell imaging and confocal microscopy in cultured hippocampal neurons, we now show that GOPs destined to major "apical" dendrites are generated from the somatic GA by a sequence of events involving: (1) generation of a GA-derived tubule; (2) tubule elongation and deployment into the dendrite; (3) tubule fission; and (4) transport and condensation of the fissioned tubule. A RhoA-Rock signaling pathway involving LIMK1, PKD1, slingshot, cofilin, and dynamin regulates polarized GOP formation by controlling the tubule fission. Our observations identify a mechanism underlying polarized GOP biogenesis and provide new insights regarding involvement of RhoA in dendritic development and polarization.

Impact of manganese on primary hippocampal neurons from rodents.

Manganese-enhanced magnetic resonance imaging (MEMRI) is a powerful tool for in vivo tract tracing or functional imaging of the central nervous system. However Mn(2+) may be toxic at high levels. In this study, we addressed the impact of Mn(2+) on mouse hippocampal neurons (HN) and neuron-like N2a cells in culture, using several approaches. Both HN and N2a cells not exposed to exogenous MnCl2 were shown by synchrotron X-ray fluorescence to contain 5 mg/g Mn. Concentrations of Mn(2+) leading to 50% lethality (LC50) after 24 h of incubation were much higher for N2a cells (863 mM) than for HN (90 mM). The distribution of Mn(2+) in both cell types exposed to Mn(2+) concentrations below LC50 was perinuclear whereas that in cells exposed to concentrations above LC50 was more diffuse, suggesting an overloading of cell storage/detoxification capacity. In addition, Mn(2+) had a cell-type and dose-dependent impact on the total amount of intracellular P, Ca, Fe and Zn measured by synchrotron X-ray fluorescence. For HN neurons, immunofluorescence studies revealed that concentrations of Mn(2+) below LC50 shortened neuritic length and decreased mitochondria velocity after 24 h of incubation. Similar concentrations of Mn(2+) also facilitated the opening of the mitochondrial permeability transition pore in isolated mitochondria from rat brains. The sensitivity of primary HN to Mn(2+) demonstrated here supports their use as a relevant model to study Mn(2+) -induced neurotoxicity.

Receptor-mediated endocytosis and trafficking between endosomal-lysosomal vacuoles in Giardia lamblia.

The early branching Giardia lamblia has highly polarized vacuoles, located underneath the plasma membrane, which have at least some of the characteristics of endosomes and of lysosomes. These peripheral vacuoles (PVs) are necessary for nutrient uptake and the maintenance of plasma membrane composition, but whether they carry out sorting and segregation of receptors and ligands is a matter of debate. Here, we showed that the internalization of low-density lipoprotein (LDL) to the PVs is highly dynamic in trophozoites with a rate similar to the internalization of the low-density lipoprotein receptor-related protein 1. Moreover, by analyzing receptor-mediated and fluid-phase endocytosis in living cells, we showed that after endocytosis LDL but not dextran moved laterally between the PVs. We speculate on PV functional heterogeneity and maturation in this parasite.

MAP6-F is a temperature sensor that directly binds to and protects microtubules from cold-induced depolymerization.

Microtubules are dynamic structures that present the peculiar characteristic to be ice-cold labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6/STOP family of proteins. However, the mechanism by which MAP6 stabilizes microtubules at 4 °C has not been identified. Moreover, the microtubule cold sensitivity and therefore the needs for microtubule stabilization in the wide range of temperatures between 4 and 37 °C are unknown. This is of importance as body temperatures of animals can drop during hibernation or torpor covering a large range of temperatures. Here, we show that in the absence of MAP6, microtubules in cells below 20 °C rapidly depolymerize in a temperature-dependent manner whereas they are stabilized in the presence of MAP6. We further show that in cells, MAP6-F binding to and stabilization of microtubules is temperature- dependent and very dynamic, suggesting a direct effect of the temperature on the formation of microtubule/MAP6 complex. We also demonstrate using purified proteins that MAP6-F binds directly to microtubules through its Mc domain. This binding is temperature-dependent and coincides with progressive conformational changes of the Mc domain as revealed by circular dichroism. Thus, MAP6 might serve as a temperature sensor adapting its conformation according to the temperature to maintain the cellular microtubule network in organisms exposed to temperature decrease.

How morphological constraints affect axonal polarity in mouse neurons.

Neuronal differentiation is under the tight control of both biochemical and physical information arising from neighboring cells and micro-environment. Here we wished to assay how external geometrical constraints applied to the cell body and/or the neurites of hippocampal neurons may modulate axonal polarization in vitro. Through the use of a panel of non-specific poly-L-lysine micropatterns, we manipulated the neuronal shape. By applying geometrical constraints on the cell body we provided evidence that centrosome location was not predictive of axonal polarization but rather follows axonal fate. When the geometrical constraints were applied to the neurites trajectories we demonstrated that axonal specification was inhibited by curved lines. Altogether these results indicated that intrinsic mechanical tensions occur during neuritic growth and that maximal tension was developed by the axon and expressed on straight trajectories. The strong inhibitory effect of curved lines on axon specification was further demonstrated by their ability to prevent formation of multiple axons normally induced by cytochalasin or taxol treatments. Finally we provided evidence that microtubules were involved in the tension-mediated axonal polarization, acting as curvature sensors during neuronal differentiation. Thus, biomechanics coupled to physical constraints might be the first level of regulation during neuronal development, primary to biochemical and guidance regulations.

Neuronal architectures with axo-dendritic polarity above silicon nanowires.

An approach is developped to gain control over the polarity of neuronal networks at the cellular level by physically constraining cell development by the use of micropatterns. It is demonstrated that the position and path of individual axons, the cell extension that propagates the neuron output signal, can be chosen with a success rate higher than 85%. This allows the design of small living computational blocks above silicon nanowires.