Cytoplasmic Dilation Research Articles

Centrosomal microtubule nucleation regulates radial migration of projection neurons independently of polarization in the developing brain

Cortical projection neurons polarize and form an axon while migrating radially. Even though these dynamic processes are closely interwoven, they are regulated separately-the neurons terminate their migration when reaching their destination, the cortical plate, but continue to grow their axons. Here, we show that in rodents, the centrosome distinguishes these processes. Newly developed molecular tools modulating centrosomal microtubule nucleation combined with invivo imaging uncovered that dysregulation of centrosomal microtubule nucleation abrogated radial migration without affecting axon formation. Tightly regulated centrosomal microtubule nucleation was required for periodic formation of the cytoplasmic dilation at the leading process, which is essential for radial migration. The microtubule nucleating factor γ-tubulin decreased at neuronal centrosomes during the migratory phase. As distinct microtubule networks drive neuronal polarization and radial migration, this provides insight into how neuronal migratory defects occur without largely affecting axonal tracts in human developmental cortical dysgeneses, caused by mutations in γ-tubulin.

Morphological and Molecular Basis of Cytoplasmic Dilation and Swelling in Cortical Migrating Neurons.

During corticogenesis, neuronal migration is an essential step for formation of a functional brain, and abnormal migration is known to cause various neurological disorders. Neuronal migration is not just a simple movement of the cell body, but a consequence of various morphological changes and coordinated subcellular events. Recent advances in in vivo and ex vivo cell biological approaches, such as in utero gene transfer, slice culture and ex vivo chemical inhibitor techniques, have revealed details of the morphological and molecular aspects of neuronal migration. Migrating neurons have been found to have a unique structure, dilation or swelling, at the proximal region of the leading process; this structure is not found in other migrating cell types. The formation of this structure is followed by nuclear deformation and forward movement, and coordination of this three-step sequential morphological change (the dilation/swelling formation, nuclear elongation and nuclear movement) is essential for proper neuronal migration and the construction of a functional brain structure. In this review, we will introduce the morphological features of this unique structure in migrating neurons and summarize what is known about the molecules regulating the dilation/swelling formation and nuclear deformation and movement.

Cdk5 and its substrates, Dcx and p27kip1, regulate the formation of cytoplasmic dilation and nuclear elongation in migrating neurons

Cdk5 and its substrates, Dcx and p27kip1, regulate the formation of cytoplasmic dilation and nuclear elongation in migrating neurons

Cdk5 and its substrates, Dcx and p27kip1, regulate cytoplasmic dilation formation and nuclear elongation in migrating neurons.

Neuronal migration is crucial for development of the mammalian-specific six-layered cerebral cortex. Migrating neurons are known to exhibit distinct features; they form a cytoplasmic dilation, a structure specific to migrating neurons, at the proximal region of the leading process, followed by nuclear elongation and forward movement. However, the molecular mechanisms of dilation formation and nuclear elongation remain unclear. Using ex vivo chemical inhibitor experiments, we show here that rottlerin, which is widely used as a specific inhibitor for PKCδ, suppresses the formation of a cytoplasmic dilation and nuclear elongation in cortical migrating neurons. Although our previous study showed that cortical neuronal migration depends on Jnk, another downstream target of rottlerin, Jnk inhibition disturbs only the nuclear elongation and forward movement, but not the dilation formation. We found that an unconventional cyclin-dependent kinase, Cdk5, is a novel downstream target of rottlerin, and that pharmacological or knockdown-mediated inhibition of Cdk5 suppresses both the dilation formation and nuclear elongation. We also show that Cdk5 inhibition perturbs endocytic trafficking as well as microtubule organization, both of which have been shown to be required for dilation formation. Furthermore, knockdown of Dcx, a Cdk5 substrate involved in microtubule organization and membrane trafficking, or p27(kip1), another Cdk5 substrate involved in actin and microtubule organization, disturbs the dilation formation and nuclear elongation. These data suggest that Cdk5 and its substrates, Dcx and p27(kip1), characterize migrating neuron-specific features, cytoplasmic dilation formation and nuclear elongation in the mouse cerebral cortex, possibly through the regulation of microtubule organization and an endocytic pathway.

Subcellular localization and function of mouse radial spoke protein 3 in mammalian cells and central nervous system.

Radial spoke protein 3 (RSP3) was first identified in Chlamydomonas as a component of the radial spoke. The mammalian homologue of the Chlamydomonas RSP3 gene is mainly expressed in testis and developing central nervous system (CNS). However, the subcellular localization and function of mammalian RSP3 in the developing brain and mammalian cells remain poorly understood. Here we show that the mouse RSP3 accumulates at the perinuclear region of Chinese hamster ovary (CHO) and 293T cells. Detailed analysis shows that the mouse RSP3 is not co-localized with the endoplasmic reticulum or Golgi apparatus markers in CHO cells. Using in utero electroporation, we found that over-expression of mammalian RSP3 increases the percentage of neurons reaching the upper cortical plate. In vivo analysis shows that the mouse RSP3 mainly accumulates in the proximal cytoplasmic dilation of the leading process of the migrating cortical neurons. Furthermore, we find that the mammalian RSP3 concentrates in the ependymal cilia as a component of the cilia. Thus, our data provide the first evidence for the subcellular localization and function of mammalian RSP3 in mammalian cells and developing CNS.

Effects of intraocular rifampicin on retinal ganglion cell structure: a stereological and histopathological study.

To determine the histopathological changes of rifampicin applied intravitreally on retinal ganglion cells by means of stereological and histopathological methods. For this study twenty-four New Zealand adult rabbits were divided into four groups (n=6 for each group). 50µg/0.1mL (group 1), 100µg/0.1mL (group 2), 150µg/0.1mL (group 3) and 200µg/0.1mL (group 4), rifampicin were injected into the vitreous of the right eyes of animals, their left eyes were used as control (group 5). After the 28(th) day of application, animals were anesthetised with xylazine (8mg/kg, IM) and then their eyes were enucleated immediately. Patterns were taken away and eyes were prepared for both stereological and electromicroscopic observation. Depending on the high dose of rifampicin, some histopathological changes such as cytoplasmic dilatation and damaged membrane were observed on the electromicroscopic level. Using quantitative examination, which was done at the light microscopic level, it was shown that the number of neurons decreased linearly as rifampicin dose increased when compared with the control group. Based on these findings, low-dose rifampicin (50µg/0.1mL) may be useful for treatment of the ocular diseases.

POSH Localizes Activated Rac1 to Control the Formation of Cytoplasmic Dilation of the Leading Process and Neuronal Migration

The formation of proximal cytoplasmic dilation in the leading process (PCDLP) of migratory neocortical neurons is crucial for somal translocation and neuronal migration, processes that require the elaborate coordination of F-actin dynamics, centrosomal movement, and nucleokinesis. However, the underlying molecular mechanisms remain poorly understood. Here, we show that the Rac1-interacting scaffold protein POSH is essential for neuronal migration in vivo. We demonstrate that POSH is concentrated in the PCDLP and that knockdown of POSH impairs PCDLP formation, centrosome translocation, and nucleokinesis. Furthermore, POSH colocalizes with F-actin and the activated form of Rac1. Knockdown of POSH impairs F-actin assembly and delocalizes activated Rac1. Interference of Rac1 activity also disrupts F-actin assembly and PCDLP formation and perturbs neuronal migration. Thus, we have uncovered a mechanism by which POSH regulates the localization of activated Rac1 and F-actin assembly to control PCDLP formation and subsequent somal translocation of migratory neurons.

Myosin II Motors and F-Actin Dynamics Drive the Coordinated Movement of the Centrosome and Soma during CNS Glial-Guided Neuronal Migration

Lamination of cortical regions of the vertebrate brain depends on glial-guided neuronal migration. The conserved polarity protein Par6alpha localizes to the centrosome and coordinates forward movement of the centrosome and soma in migrating neurons. The cytoskeletal components that produce this unique form of cell polarity and their relationship to polarity signaling cascades are unknown. We show that F-actin and Myosin II motors are enriched in the neuronal leading process and that Myosin II activity is necessary for leading process actin dynamics. Inhibition of Myosin II decreased the speed of centrosome and somal movement, whereas Myosin II activation increased coordinated movement. Ectopic expression or silencing of Par6alpha inhibited Myosin II motors by decreasing Myosin light-chain phosphorylation. These findings suggest leading-process Myosin II may function to "pull" the centrosome and soma forward during glial-guided migration by a mechanism involving the conserved polarity protein Par6alpha.

Cytoskeletal coordination during neuronal migration

Discoveries from human and mouse genetics have identified cytoskeletal and signaling proteins that are essential for neuronal migration in the developing brain. To provide a meaningful context for these studies, we took an unbiased approach of correlative electron microscopy of neurons migrating through a three-dimensional matrix, and characterized the cytoskeletal events that occur as migrating neurons initiate saltatory forward movements of the cell nucleus. The formation of a cytoplasmic dilation in the proximal leading process precedes nuclear translocation. Cell nuclei translocate into these dilations in saltatory movements. Time-lapse imaging and pharmacological perturbation suggest that nucleokinesis requires stepwise or hierarchical interactions between microtubules, myosin II, and cell adhesion. We hypothesize that these interactions couple leading process extension to nuclear translocation during neuronal migration.

Delayed neuronal recovery and neuronal death in rat hippocampus following severe cerebral ischemia: possible relationship to abnormalities in neuronal processes.

Mechanisms involved in the postischemic delay in neuronal recovery or death in rat hippocampus were evaluated by light and electron microscopy at 3, 15, 30, and 120 min and 24, 36, 48, and 72 h following severe cerebral ischemia that was produced by permanent occlusion of the vertebral arteries and 30-min occlusion of the common carotid arteries. During the early postischemic period, neurons in the Ca1 and Ca3 regions both showed transient mitochondrial swelling followed by the disaggregation of polyribosomes, decrease in rough endoplasmic reticulum (RER), loss of Golgi apparatus (GA) cisterns, and decrease in GA vesicles . Recovery of these organelles in Ca3 neurons was first noted between 24 and 36 h and was accompanied by a marked proliferation of smooth endoplasmic reticulum (SER). Many Ca1 neurons initially recovered between 24 and 36 h, but subsequent cell death at 48-72 h was often preceded by peripheral chromatolysis, constriction and shrinkage of the proximal dendrites, and cytoplasmic dilatation that was continuous with focal expansion of RER cisterns. Because SER accumulates in resistant Ca3 neurons and proximal neuronal processes are damaged in vulnerable Ca1 neurons, we hypothesize that delayed cell recovery or death in vulnerable and resistant postischemic hippocampal neurons is related to abnormalities in neuronal processes.