Cholesterol-dependent modulation of dendrite outgrowth and microtubule stability in cultured neurons

Microtubule-associated protein 2 (MAP2) is a neuron-specific cytoskeletal protein enriched in dendrites and cell bodies. MAP2 regulates microtubule stability in a phosphorylation-dependent manner, which has been implicated in dendrite outgrowth and branching. We have previously reported that cholesterol deficiency causes tau phosphorylation and microtubule depolymerization in axons (Fan et al. 2001). To investigate whether cholesterol also modulates microtubule stability in dendrites by modulating MAP2 phosphorylation, we examined the effect of compactin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and TU-2078 (TU), a squalene epoxidase inhibitor, on these parameters using cultured neurons. We have found that cholesterol deficiency induced by compactin and TU, inhibited dendrite outgrowth, but not of axons, and attenuated axonal branching. Dephosphorylation of MAP2 and microtubule depolymerization accompanied these alterations. The amount of protein phosphatase 2 A (PP2A) and its activity in association with microtubules were decreased, while those unbound to microtubules were increased. The synthesized ceramide levels and the total ceramide content were increased in these cholesterol-deficient neurons. These alterations caused by compactin were prevented by concurrent treatment of cultured neurons with beta-migrating very-low-density lipoproteins (beta-VLDL) or cholesterol. Taken together, we propose that cholesterol-deficiency causes a selective inhibition of dendrite outgrowth due to the decreased stability of microtubules as a result of inhibition of MAP2 phosphorylation.     

A Highlights from MBoC Selection: γ-Tubulin controls neuronal microtubule polarity independently of Golgi outposts

Neurons have highly polarized arrangements of microtubules, but it is incompletely understood how microtubule polarity is controlled in either axons or dendrites. To explore whether microtubule nucleation by γ-tubulin might contribute to polarity, we analyzed neuronal microtubules in Drosophila containing gain- or loss-of-function alleles of γ-tubulin. Both increased and decreased activity of γ-tubulin, the core microtubule nucleation protein, altered microtubule polarity in axons and dendrites, suggesting a close link between regulation of nucleation and polarity. To test whether nucleation might locally regulate polarity in axons and dendrites, we examined the distribution of γ-tubulin. Consistent with local nucleation, tagged and endogenous γ-tubulins were found in specific positions in dendrites and axons. Because the Golgi complex can house nucleation sites, we explored whether microtubule nucleation might occur at dendritic Golgi outposts. However, distinct Golgi outposts were not present in all dendrites that required regulated nucleation for polarity. Moreover, when we dragged the Golgi out of dendrites with an activated kinesin, γ-tubulin remained in dendrites. We conclude that regulated microtubule nucleation controls neuronal microtubule polarity but that the Golgi complex is not directly involved in housing nucleation sites.     

The FTLD risk factor TMEM106B and MAP6 control dendritic trafficking of lysosomes

TMEM106B is a major risk factor for frontotemporal lobar degeneration with TDP‐43 pathology. TMEM106B localizes to lysosomes, but its function remains unclear. We show that TMEM106B knockdown in primary neurons affects lysosomal trafficking and blunts dendritic arborization. We identify microtubule‐associated protein 6 (MAP6) as novel interacting protein for TMEM106B. MAP6 over‐expression inhibits dendritic branching similar to TMEM106B knockdown. MAP6 knockdown fully rescues the dendritic phenotype of TMEM106B knockdown, supporting a functional interaction between TMEM106B and MAP6. Live imaging reveals that TMEM106B knockdown and MAP6 overexpression strongly increase retrograde transport of lysosomes in dendrites. Downregulation of MAP6 in TMEM106B knockdown neurons restores the balance of anterograde and retrograde lysosomal transport and thereby prevents loss of dendrites. To strengthen the link, we enhanced anterograde lysosomal transport by expressing dominant‐negative Rab7‐interacting lysosomal protein (RILP), which also rescues the dendrite loss in TMEM106B knockdown neurons. Thus, TMEM106B/MAP6 interaction is crucial for controlling dendritic trafficking of lysosomes, presumably by acting as a molecular brake for retrograde transport. Lysosomal misrouting may promote neurodegeneration in patients with TMEM106B risk variants.     

CNP/cGMP signaling regulates axon branching and growth by modulating microtubule polymerization

The peptide hormone CNP has recently been found to positively regulate axon branching and growth via activation of cGMP signaling in embryonic dorsal root ganglion (DRG) neurons, but the cellular mechanisms mediating the regulation of these developmental processes have not been established. In this study, we provide evidence linking CNP/cGMP signaling to microtubule dynamics via the microtubule regulator CRMP2. First, phosphorylation of CRMP2 can be suppressed by cGMP activation in embryonic DRG neurons, and non‐phosphorylated CRMP2 promotes axon branching and growth. In addition, real time analysis of growing microtubule ends indicates a similar correlation of CRMP2 phosphorylation and its activity in promoting microtubule polymerization rates and durations in both COS cells and DRG neuron growth cones. Moreover, direct activation of cGMP signaling leads to increased assembly of dynamic microtubules in DRG growth cones. Finally, low doses of a microtubule depolymerization drug nocodazole block CNP/cGMP‐dependent axon branching and growth. Taken together, our results support a critical role of microtubule dynamics in mediating CNP/cGMP regulation of axonal development. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 673–687, 2013     

More than just synaptic building blocks: scaffolding proteins of the post-synaptic density regulate dendritic patterning

The dendritic arbor is responsible for receiving and consolidating neuronal input. Outgrowth and morphogenesis of the arbor are complex stages of development that are poorly understood. However, recent findings have identified synaptic scaffolding proteins as novel regulators of these important events. Scaffolding proteins are enriched in the post-synaptic density where they bind and bring into close proximity neurotransmitter receptors, signaling molecules, and regulators of the actin cytoskeleton. This property is important for dendritic spine morphogenesis and maintenance in the mature neuron. Scaffolding proteins are now being described as key regulators of neurite outgrowth, dendritic development, and pattern formation in immature neurons. These proteins, which include post-synaptic-95, Shank and Densin-180, as well as many of their interacting partners, appear to regulate both the microtubule and actin cytoskeleton to influence dendrite morphology. Through a large array of protein-protein interaction domains, scaffolding proteins are able to form large macromolecular complexes that include cytoskeletal motor proteins as well as microtubule and actin regulatory molecules. Together, the new findings form a persuasive argument that scaffolding proteins deliver critical regulatory elements to sites of dendritic outgrowth and branching to modulate the formation and maintenance of the dendritic arbor.     

Cellular mechanisms of dendrite pruning in Drosophila: insights from in vivo time-lapse of remodeling dendritic arborizing sensory neurons

Regressive events that refine exuberant or inaccurate connections are critical in neuronal development. We used multi-photon, time-lapse imaging to examine how dendrites of Drosophila dendritic arborizing (da) sensory neurons are eliminated during early metamorphosis, and how intrinsic and extrinsic cellular mechanisms control this deconstruction. Removal of the larval dendritic arbor involves two mechanisms: local degeneration and branch retraction. In local degeneration, major branch severing events entail focal disruption of the microtubule cytoskeleton, followed by thinning of the disrupted region, severing and fragmentation. Retraction was observed at distal tips of branches and in proximal stumps after severing events. The pruning program of da neuron dendrites is steroid induced; cell-autonomous dominant-negative inhibition of steroid action blocks local degeneration, although retraction events still occur. Our data suggest that steroid-induced changes in the epidermis may contribute to dendritic retraction. Finally, we find that phagocytic blood cells not only engulf neuronal debris but also attack and sever intact branches that show signs of destabilization.     

Dynamics of outgrowth in a continuum model of neurite elongation

Neurite outgrowth (dendrites and axons) should be a stable, but easily regulated process to enable a neuron to make its appropriate network connections during development. We explore the dynamics of outgrowth in a mathematical continuum model of neurite elongation. The model describes the construction of the internal microtubule cytoskeleton, which results from the production and transport of tubulin dimers and their assembly into microtubules at the growing neurite tip. Tubulin is assumed to be largely synthesised in the cell body from where it is transported by active mechanisms and by diffusion along the neurite. It is argued that this construction process is a fundamental limiting factor in neurite elongation. In the model, elongation is highly stable when tubulin transport is dominated by either active transport or diffusion, but oscillations in length may occur when both active transport and diffusion contribute. Autoregulation of tubulin production can eliminate these oscillations. In all cases a stable steady-state length is reached, provided there is intrinsic decay of tubulin. Small changes in growth parameters, such as the tubulin production rate, can lead to large changes in length. Thus cytoskeleton construction can be both stable and easily regulated, as seems necessary for neurite outgrowth during nervous system development. Action Editor: Upinder Bhalla     

Neural activity and branching of embryonic retinal ganglion cell dendrites

The shape of a neuron's dendritic arbor is critical for its function as it determines the number of inputs the neuron can receive and how those inputs are processed. During development, a neuron initiates primary dendrites that branch to form a simple arbor. Subsequently, growth occurs by a process that combines the extension and retraction of existing dendrites, and the addition of new branches. The loss and addition of the fine terminal branches of retinal ganglion cells (RGCs) is dependent on afferent inputs from its synaptic partners, the amacrine and bipolar cells. It is unknown, however, whether neural activity regulates the initiation of primary dendrites and their initial branching. To investigate this, Xenopus laevis RGCs developing in vivo were made to express either a delayed rectifier type voltage-gated potassium (KV) channel, Xenopus Kv1.1, or a human inward rectifying channel, Kir2.1, shown previously to modulate the electrical activity of Xenopus spinal cord neurons. Misexpression of either potassium channel increased the number of branch points and the total length of all the branches. As a result, the total dendritic arbor was bigger than for control green fluorescent protein-expressing RGCs and those ectopically expressing a highly related mutant non-functional Kv1.1 channel. Our data indicate that membrane excitability regulates the earliest differentiation of RGC dendritic arbors.     

Differential regulation of dendrite complexity by AMPA receptor subunits GluR1 and GluR2 in motor neurons

Activity-dependent developmental mechanisms in many regions of the central nervous system are thought to be responsible for shaping dendritic architecture and connectivity, although the molecular mechanisms underlying these events remain obscure. Since AMPA glutamate receptors are developmentally regulated in spinal motor neurons, we have investigated the role of activation of AMPA receptors in dendritic outgrowth of spinal motor neurons by overexpression of two subunits, GluR1 and GluR2, and find that dendrite outgrowth is differentially controlled by expression of these subunits. Overexpression of GluR1 was associated with greater numbers of filopodia, and an increase in the length and complexity of dendritic arbor. In contrast, GluR2 expression did not alter dendritic complexity, but was associated with a moderate increase in length of arbor, and decreased numbers of filopodia. Neither GluR1 nor GluR2 had any effect on the motility of filopodia. In addition, GluR1 but not GluR2 expression increased the density of dendritic puncta incorporating a GFP-labeled PSD95, suggesting that GluR1 may mediate its effect in part by augmenting the number of excitatory synapses within motor neuron dendrites. Together these results suggest that in spinal motor neurons, AMPA receptors composed of GluR1 subunits may facilitate neurotrophic mechanisms in these neurons, permitting sustained dendrite outgrowth and synaptogenesis, whereas expression of AMPA receptors containing GluR2 acts to preserve existing dendritic arbor. Thus, the observed downregulation of GluR1 in motor neurons during postnatal development may limit the formation of new dendrite segments and synapses, promoting stabilized synaptic connectivity.     

Method of modelling intracellular transport in branching neurites: application to axons and dendrites of Drosophila sensory neurons

This paper develops a method of calculating the transport of intracellular organelles in neurons with branching neurites which is based on the Smith–Simmons equations of motor-assisted transport. The method is aimed at understanding the effects of microtubule (MT) polarity orientation in branching neurites on transport of organelles at the fundamental level. The method is applied to calculating the organelle transport in axons and dendrites of Drosophila neurons, using the map of MT orientation in such neurons developed by Stone et al. (Mol Biol Cell 19:4122–4129, 2008). The proximal dendrite is assumed to branch and form two distal dendrites. Two different MT polarity arrangements in a proximal dendrite are considered, and implications of these MT arrangements on organelle transport are analysed. It is demonstrated that the MT arrangement found in Drosophila dendrites (MTs have their minus ends out in a proximal dendrite) results in much more efficient motor-driven transport than the structure with a mixed MT orientation in proximal dendrites.     

RAB-10 Regulates Dendritic Branching by Balancing Dendritic Transport

The construction of a large dendritic arbor requires robust growth and the precise delivery of membrane and protein cargoes to specific subcellular regions of the developing dendrite. How the microtubule-based vesicular trafficking and sorting systems are regulated to distribute these dendritic development factors throughout the dendrite is not well understood. Here we identify the small GTPase RAB-10 and the exocyst complex as critical regulators of dendrite morphogenesis and patterning in the C. elegans sensory neuron PVD. In rab-10 mutants, PVD dendritic branches are reduced in the posterior region of the cell but are excessive in the distal anterior region of the cell. We also demonstrate that the dendritic branch distribution within PVD depends on the balance between the molecular motors kinesin-1/UNC-116 and dynein, and we propose that RAB-10 regulates dendrite morphology by balancing the activity of these motors to appropriately distribute branching factors, including the transmembrane receptor DMA-1.     

Interaction of spindle assembly factor TPX2 with importins-α/β inhibits protein phase separation

The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed via several microtubule nucleation pathways. Targeting Protein for XKlp2 (TPX2) stimulates the branching microtubule nucleation pathway, where new microtubules are nucleated from preexisting ones within mitotic or meiotic spindles. TPX2, like other spindle assembly factors, is sequestered by binding to nuclear importins-α/β until the onset of mitosis, yet the molecular nature of this regulation remains unclear. Here we demonstrate that TPX2 interacts with importins-α/β with nanomolar affinity in a 1:1:1 monodispersed trimer. We also identify a new nuclear localization sequence in TPX2 that contributes to its high-affinity interaction with importin-α. In addition, we establish that TPX2 interacts with importin-β via dispersed, weak interactions. We show that interactions of both importin-α and -β with TPX2 inhibit its ability to undergo phase separation, which was recently shown to enhance the kinetics of branching microtubule nucleation. In summary, our study informs how importins regulate TPX2 to facilitate spindle assembly, and provides novel insight into the functional regulation of protein phase separation.     

Modeling organelle transport in branching dendrites with a variable cross-sectional area

The purpose of this paper is to develop a method for calculating organelle transport in dendrites with a non-uniform cross-sectional area that depends on the distance from the neuron soma. The model is based on modified Smith–Simmons equations governing molecular motor-assisted organelle transport. The developed method is then applied to simulating organelle transport in branching dendrites with two particular microtubule (MT) orientations reported from experiments. It is found that the rate of organelle transport toward a dendrite’s growth cone heavily depends on the MT orientation, and since there is experimental evidence that the MT orientation in a particular region of a dendrite may depend on the dendrite’s developmental stage, the obtained results suggest that a rearrangement of the MT structure may depend on the amount of organelles needed at the growth cone.     

Sporoptosis: sowing the seeds of nuclear destruction

Previous models of neuronal dendrite arborization suggested that contact-dependent self-avoidance between dendrite branches prevents self-crossings within the arbor. Two papers in Neuron show how integrin-mediated adhesion to the extracellular matrix restricts dendrites to a two-dimensional space to optimize this mechanism (Han et al., 2012; Kim et al., 2012).     

Differentiation of Apical and Basal Dendrites in Pyramidal Cells and Granule Cells in Dissociated Hippocampal Cultures

Hippocampal pyramidal cells and dentate granule cells develop morphologically distinct dendritic arbors, yet also share some common features. Both cell types form a long apical dendrite which extends from the apex of the cell soma, while short basal dendrites are developed only in pyramidal cells. Using quantitative morphometric analyses of mouse hippocampal cultures, we evaluated the differences in dendritic arborization patterns between pyramidal and granule cells. Furthermore, we observed and described the final apical dendrite determination during dendritic polarization by time-lapse imaging. Pyramidal and granule cells in culture exhibited similar dendritic patterns with a single principal dendrite and several minor dendrites so that the cell types were not readily distinguished by appearance. While basal dendrites in granule cells are normally degraded by adulthood in vivo, cultured granule cells retained their minor dendrites. Asymmetric growth of a single principal dendrite harboring the Golgi was observed in both cell types soon after the onset of dendritic growth. Time-lapse imaging revealed that up until the second week in culture, final principal dendrite designation was not stabilized, but was frequently replaced by other minor dendrites. Before dendritic polarity was stabilized, the Golgi moved dynamically within the soma and was repeatedly repositioned at newly emerging principal dendrites. Our results suggest that polarized growth of the apical dendrite is regulated by cell intrinsic programs, while regression of basal dendrites requires cue(s) from the extracellular environment in the dentate gyrus. The apical dendrite designation is determined from among multiple growing dendrites of young developing neurons.     

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites,Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis¹, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation^2-5 and degeneration⁶.The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron^7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape^4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring ^12,13.In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.Download video file.^{(47M, mov)}     

Neuronal types in the striatum of man

Summary Nerve cells of the human striatum were investigated with the use of a newly developed technique that reveals the pattern of pigmentation of individual nerve cells by means of transparent Golgi impregnations of their cell bodies and processes. Five types of neurons are distinguished:Type I is a medium-sized spine-laden neuron with an axon giving off a great number of collateral branches. The vast majority of the cells in the striatum belong to this type. Numerous intensely stained lipofuscin granules are contained in one pole of the cell body and may also extend into adjacent portions of a dendrite.Type II is a medium-sized to large neuron with long intertwining dendrites decorated with spines of uncommon shape. A distinguishing feature of this cell type is the presence of somal spines. This cell type is devoid of pigment or contains only a few tiny lipofuscin granules.Type III is a large multipolar neuron. The cell body generates a few rather extended dendrites that are very sparsely spined. The finely granulated pigment is evenly dispersed within a large portion of the cytoplasm.Type IV is a large aspiny neuron with rounded cell body and richly branching tortuous dendrites. The axon branches frequently in the vicinity of the parent soma. Large pigment granules are concentrated within a circumscribed part of the cell body close to the cell membrane.Type V is a small to medium-sized aspiny neuron. The dendrites break up into a swirling mass of thin branches. More than one axon may be given off from the soma. The axons branch close to the soma into terminal twigs. Cells of this type contain numerous large and well-stained lipofuscin granules.Each of the cell types has a characteristic pattern of pigmentation. The different varieties of nerve cells in the striatum can therefore be distinguished not only in Golgi impregnations but also in pigment-Nissl preparations.     

Organization and Control of Microtubule Pattern in Centrohelidan Heliozoa*†

SYNOPSIS. Comparative studies of axopodial microtubule pattern in 10 different centrohelidan Heliozoa belonging to the genera Acanthocystis, Raphidiophrys and Heterophrys suggest that 2 basic principles govern pattern formation in centrohelidan Heliozoa. While the larger “open” arrays with unspecified number of microtubules, e.g. in A. aculeata and R. ambigua, may result from self-linkage of additional microtubules around centroplast-nucleated “starter microtubules,” the smaller “closed” arrays with specified microtubule number, e.g. in A. pectinata and H. marina, favor a template-driven linkage mechanism. The centroplast is a highly complex microtubule organizing center involved in the control of orientation, number, and diameter of the axonemes. Its shell may serve as a surface upon which the microtubule nucleating sites assemble, but how the precise positioning of these sites occurs is still open to debate. Some of the unsolved problems of microtubule pattern formation may be explained by the “linker nucleation hypothesis” which is an extension of the “gradion hypothesis” by Roth et al. It is shown how both the formation of closed arrays and the balanced lateral growth of open arrays may result from linker-induced microtubule nucleation.