in-vivo-postnatal-electroporation-time-lapse-imaging-neuroblast

<em>In vivo</em> Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

nucleofection-rodent-neuroblasts-to-study-neuroblast-migration

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration <em>In vitro</em>

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

King's College London

Axonal retrograde transport is essential for neuronal growth and survival. However, the nature and dynamics of the membrane compartments involved in this process are poorly characterized. To shed light on this pathway, we established an experimental system for the visualization and the quantitative study of retrograde transport in living motor neurons based on a fluorescent fragment of tetanus toxin (TeNT HC). Morphological and kinetic analysis of TeNT HC retrograde carriers reveals two major groups of organelles: round vesicles and fast tubular structures. TeNT HC carriers lack markers of the classical endocytic pathway and are not acidified during axonal transport. Importantly, TeNT HC and NGF share the same retrograde transport organelles, which are characterized by the presence of the neurotrophin receptor p75NTR. Our results provide the first direct visualization of retrograde transport in living motor neurons, and reveal a novel retrograde route that could be used both by physiological ligands (i.e., neurotrophins) and TeNT to enter the central nervous system.

Analysis of retrograde transport in motor neurons reveals common endocytic carriers for tetanus toxin and neurotrophin receptor p75NTR.

Degenerative disorders of motor neurons include a range of progressive fatal diseases such as amyotrophic lateral sclerosis (ALS), spinal-bulbar muscular atrophy (SBMA), and spinal muscular atrophy (SMA). Although the causative genetic alterations are known for some cases, the molecular basis of many SMA and SBMA-like syndromes and most ALS cases is unknown. Here we show that missense point mutations in the cytoplasmic dynein heavy chain result in progressive motor neuron degeneration in heterozygous mice, and in homozygotes this is accompanied by the formation of Lewy-like inclusion bodies, thus resembling key features of human pathology. These mutations exclusively perturb neuron-specific functions of dynein.

Mutations in dynein link motor neuron degeneration to defects in retrograde transport.

Anaerobic bacteria of the genus Clostridia are a major threat to human and animal health, being responsible for pathologies ranging from food poisoning to gas gangrene. In each of these, the production of sophisticated exotoxins is the main cause of disease. The most powerful clostridial toxins are tetanus and botulinum neurotoxins, the causative agents of tetanus and botulism. They are structurally organized into three domains endowed with distinct functions: high affinity binding to neurons, membrane translocation and specific cleavage of proteins controlling neuroexocytosis. Recent discoveries regarding the mechanism of membrane recruitment and sorting of these neurotoxins within neurons make them ideal tools to uncover essential aspects of neuronal physiology in health and disease.

The journey of tetanus and botulinum neurotoxins in neurons.

Using a novel assay based on the sorting and transport of a fluorescent fragment of tetanus toxin, we have investigated the cytoskeletal and motor requirements of axonal retrograde transport in living mammalian motor neurons. This essential process ensures the movement of neurotrophins and organelles from the periphery to the cell body and is crucial for neuronal survival. Unlike what is observed in sympathetic neurons, fast retrograde transport in motor neurons requires not only intact microtubules, but also actin microfilaments. Here, we show that the movement of tetanus toxin-containing carriers relies on the nonredundant activities of dynein as well as kinesin family members. Quantitative kinetic analysis indicates a role for dynein as the main motor of these carriers. Moreover, this approach suggests the involvement of myosin(s) in retrograde movement. Immunofluorescence screening with isoform-specific myosin antibodies reveals colocalization of tetanus toxin-containing retrograde carriers with myosin Va. Motor neurons from homozygous myosin Va null mice showed slower retrograde transport compared with wild-type cells, establishing a unique role for myosin Va in this process. On the basis of our findings, we propose that coordination of myosin Va and microtubule-dependent motors is required for fast axonal retrograde transport in motor neurons.

Myosin Va and microtubule-based motors are required for fast axonal retrograde transport of tetanus toxin in motor neurons.

Neurite branching is essential for the establishment of appropriate neuronal connections during development and regeneration. We identify the small GTPase Ral as a mediator of neurite branching. Active Ral promotes neurite branching in cortical and sympathetic neurons, whereas Ral inhibition decreases laminin-induced branching. In addition, depletion of endogenous Ral by RNA interference decreases branching in cortical neurons. The two Ral isoforms, RalA and -B, promote branching through distinct pathways, involving the exocyst complex and phospholipase D, respectively. Finally, Ral-dependent branching is mediated by protein kinase C-dependent phosphorylation of 43-kD growth-associated protein, a crucial molecule involved in pathfinding, plasticity, and regeneration. These findings highlight an important role for Ral in the regulation of neuronal morphology.

Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex.

Efficient gene transfer is an important tool for the study of neuronal function and biology. This has proved difficult and inefficient with traditional transfection strategies, which can also be fairly toxic, whereas viral-mediated gene transfer, although highly efficient, is often time-consuming. The recently developed Amaxa Nucleofector technology, based on electroporation with preset parameters in a cell-type-specific solution, enables direct delivery of DNA, small interfering (si)RNA oligonucleotides and siRNA vectors into the cell nucleus. This strategy results in reproducible, rapid, and efficient transfection of a broad range of cells, including primary neurons. Nucleofected neurons survive for up to 3 weeks and remain functional. We are currently using this transfection method to examine the contribution of Rho GTPase signaling pathways in the establishment of neuronal polarity, neuronal migration, and neurite outgrowth. Here, we describe three protocols to efficiently nucleofect rat cerebellar granule, cortical, and hippocampal neurons.

Nucleofection of primary neurons.

Neuronal polarization requires localized cytoskeletal changes and polarized membrane traffic. Here, I report that the small GTPase RalA, previously shown to control neurite branching, also regulates neuronal polarity. RalA depletion, or ectopic expression of constitutively active RalA in cultured neurons inhibit axon formation. However, expression of a constitutively active RalA mutant that is unable to interact with the exocyst complex has no effect on neuronal polarization. Furthermore, depletion of the Sec6, Sec8 or Exo84 subunits of the exocyst complex also leads to unpolarized neurons. Early stages of neuronal polarization are accompanied by increasing levels of interaction of the exocyst complex with PAR-3 and atypical protein kinase C (aPKC), and by the RalA-dependent association of the exocyst complex with PAR-3. Thus, neuronal polarization involves a RalA-regulated association between mediators of vesicle trafficking (exocyst complex) and cell polarity (PAR-3).

RalA and the exocyst complex influence neuronal polarity through PAR-3 and aPKC.

The establishment of precise neuronal cell morphology provides the foundation for all aspects of neurobiology. During development, axons emerge from cell bodies after an initial polarization stage, elongate, and navigate towards target regions guided by a range of environmental cues. The Rho and Ras families of small GTPases have emerged as critical players at all stages of axonogenesis. Their ability to coordinately direct multiple signal transduction pathways with precise spatial control drives many of the activities that underlie this morphogenetic program: the dynamic assembly, disassembly, and reorganization of the actin and microtubule cytoskeletons, the interaction of the growing axon with other cells and extracellular matrix, the delivery of lipids and proteins to the axon through the exocytic machinery, and the internalization of membrane and proteins at the leading edge of the growth cone through endocytosis. This article highlights the contribution of Rho and Ras GTPases to axonogenesis.

Rho and Ras GTPases in axon growth, guidance, and branching.

In the adult brain, neural stem cells proliferate within the subventricular zone before differentiating into migratory neuroblasts that travel along the rostral migratory stream (RMS) to populate the olfactory bulb with new neurons. Because neuroblasts have been shown to migrate to areas of brain injury, understanding the cues regulating this migration could be important for brain repair. Recent studies have highlighted an important role for endocannabinoid (eCB) signaling in the proliferation of the stem cell population, but it remained to be determined whether this pathway also played a role in cell migration. We now show that mouse migratory neuroblasts express cannabinoid receptors, diacylglycerol lipase α (DAGLα), the enzyme that synthesizes the endocannabinoid 2-arachidonoylglycerol (2-AG), and monoacylglycerol lipase, the enzyme responsible for its degradation. Using a scratch wound assay for a neural stem cell line and RMS explant cultures, we show that inhibition of DAGL activity or CB(1)/CB(2) receptors substantially decreases migration. In contrast, direct activation of cannabinoid receptors or preventing the breakdown of 2-AG increases migration. Detailed analysis of primary neuroblast migration by time-lapse imaging reveals that nucleokinesis, as well as the length and branching of the migratory processes are under dynamic control of the eCB system. Finally, similar effects are observed in vivo by analyzing the morphology of green fluorescent protein-labeled neuroblasts in brain slices from mice treated with CB(1) or CB(2) antagonists. These results describe a novel role for the endocannabinoid system in neuroblast migration in vivo, highlighting its importance in regulating an additional essential step in adult neurogenesis.

Endocannabinoids regulate the migration of subventricular zone-derived neuroblasts in the postnatal brain.

Cell polarization is critical for the correct functioning of many cell types, creating functional and morphological asymmetry in response to intrinsic and extrinsic cues. Neurons are a classical example of polarized cells, as they usually extend one long axon and short branched dendrites. The formation of such distinct cellular compartments (also known as neuronal polarization) ensures the proper development and physiology of the nervous system and is controlled by a complex set of signalling pathways able to integrate multiple polarity cues. Because polarization is at the basis of neuronal development, investigating the mechanisms responsible for this process is fundamental not only to understand how the nervous system develops, but also to devise therapeutic strategies for neuroregeneration. The last two decades have seen remarkable progress in understanding the molecular mechanisms responsible for mammalian neuronal polarization, primarily using cultures of rodent hippocampal neurons. More recent efforts have started to explore the role of such mechanisms in vivo. It has become clear that neuronal polarization relies on signalling networks and feedback mechanisms co-ordinating the actin and microtubule cytoskeleton and membrane traffic. The present chapter will highlight the role of key molecules involved in neuronal polarization, such as regulators of the actin/microtubule cytoskeleton and membrane traffic, polarity complexes and small GTPases.

Crucial polarity regulators in axon specification.

After birth, stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). This migration is a fundamental event controlling the proper integration of new neurons in a pre-existing synaptic network. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. Here, we show that the actin-bundling protein fascin is highly upregulated in mouse SVZ-derived migratory neuroblasts. Fascin-1ko mice display an abnormal RMS and a smaller OB. Bromodeoxyuridine labeling experiments show that lack of fascin significantly impairs neuroblast migration, but does not appear to affect cell proliferation. Moreover, fascin depletion substantially alters the polarized morphology of rat neuroblasts. Protein kinase C (PKC)-dependent phosphorylation of fascin on Ser39 regulates its actin-bundling activity. In vivo postnatal electroporation of phosphomimetic (S39D) or nonphosphorylatable (S39A) fascin variants followed by time-lapse imaging of brain slices demonstrates that the phospho-dependent modulation of fascin activity ensures efficient neuroblast migration. Finally, fluorescence lifetime imaging microscopy studies in rat neuroblasts reveal that the interaction between fascin and PKC can be modulated by cannabinoid signaling, which controls neuroblast migration in vivo. We conclude that fascin, whose upregulation appears to mark the transition to the migratory neuroblast stage, is a crucial regulator of neuroblast motility. We propose that a tightly regulated phospho/dephospho-fascin cycle modulated by extracellular signals is required for the polarized morphology and migration in neuroblasts, thus contributing to efficient neurogenesis.

Giovanna Lalli

Wolfson Centre for Age-Related Diseases

King's College London

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Giovanna Lalli

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King's College London

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Wolfson Centre for Age-Related Diseases