Reeler: new tales on an old mutant mouse

Neurological mouse mutants provide an opportunity to dissect the complex mechanisms that underlie vertebrate brain development. Advances in genetic technologies have permitted the identification of genes disrupted in many mutants, allowing a molecular interpretation of the phenotypes. For several decades, the spontaneous mutant mouse reeler has been used as a model for the analysis of the development of laminated brain structures. In this ataxic mutant, the migration of many neurons is aberrant, resulting in disrupted cellular organization. Recently, reelin, the gene disrupted in the reeler mouse, has been identified. reelin encodes a novel extracellular molecule that controls neural cell positioning through mechanisms that are not yet completely understood. Analysis of the expression pattern and the properties of the reelin gene product (Reelin) suggests models for its function during brain development. Furthermore, the recent identification of genes that may function in the Reelin signaling pathway advances our knowledge of the molecular basis of neuronal migration. BioEssays 20 :235–244, 1998.© 1998 John Wiley & Sons, Inc.     

Cellular migration and axonal outgrowth from adult mammalian peripheral nerves in vitro

It is known that following peripheral nerve transections, sheath cells proliferate and migrate to form a bridge between nerve stumps, which may facilitate axonal regeneration. In the present investigations, cellular migration and axonal outgrowth from nerves of adult mice were studied in vitro using collagen gels. During the first 3 days in culture, profuse migration of fibroblasts and macrophages occurred from the ends of sciatic nerve segments, which had been lesioned in situ a few days prior to explanation, but not from segments of normal nerves. The mechanism of cellular activation in the lesioned nerves was not determined, but migration was blocked by suramin, which inhibits the actions of several growth factors. The migrating cells, which form the bridge tissue, may promote axonal regeneration in two ways. Firstly, axonal outgrowth from isolated intercostal nerves was significantly increased in co-cultures with bridges from lesioned sciatic nerves. This stimulatory effect was inhibited by antibodies to 2.5S nerve growth factor. Secondly, the segments of bridge tissue contracted when removed from animals. It is possible that fibroblasts within the bridge exert traction that would tend to pull the lesioned stumps of peripheral nerve together, as in the healing of skin wounds. The traction may also influence deposition of extracellular matrix materials, such as collagen fibrils, which could orient the growth of the regenerating axons toward the distal nerve stump. © 1996 John Wiley & Sons, Inc.     

Alterations of autophagy in the peripheral neuropathy Charcot-Marie-Tooth type 2B

Charcot-Marie-Tooth type 2B (CMT2B) disease is a dominant axonal peripheral neuropathy caused by 5 mutations in the RAB7A gene, a ubiquitously expressed GTPase controlling late endocytic trafficking. In neurons, RAB7A also controls neuronal-specific processes such as NTF (neurotrophin) trafficking and signaling, neurite outgrowth and neuronal migration. Given the involvement of macroautophagy/autophagy in several neurodegenerative diseases and considering that RAB7A is fundamental for autophagosome maturation, we investigated whether CMT2B-causing mutants affect the ability of this gene to regulate autophagy. In HeLa cells, we observed a reduced localization of all CMT2B-causing RAB7A mutants on autophagic compartments. Furthermore, compared to expression of RAB7A^WT, expression of these mutants caused a reduced autophagic flux, similar to what happens in cells expressing the dominant negative RAB7A^T22N mutant. Consistently, both basal and starvation-induced autophagy were strongly inhibited in skin fibroblasts from a CMT2B patient carrying the RAB7A^V162M mutation, suggesting that alteration of the autophagic flux could be responsible for neurodegeneration.     

Modified cell cycle status in a mouse model of altered neuronal vulnerability (slow Wallerian degeneration; <Emphasis Type="Italic">Wld</Emphasis><Superscript><Emphasis Type="Italic">s</Emphasis></Superscript>)

Background  

Altered neuronal vulnerability underlies many diseases of the human nervous system, resulting in degeneration and loss of neurons. The neuroprotective slow Wallerian degeneration (Wld ^s ) mutation delays degeneration in axonal and synaptic compartments of neurons following a wide range of traumatic and disease-inducing stimuli, providing a powerful experimental tool with which to investigate modulation of neuronal vulnerability. Although the mechanisms through which Wld ^s confers neuroprotection remain unclear, a diverse range of downstream modifications, incorporating several genes/pathways, have been implicated. These include the following: elevated nicotinamide adenine dinucleotide (NAD) levels associated with nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1; a part of the chimeric Wld ^s gene); altered mRNA expression levels of genes such as pituitary tumor transforming gene 1 (Pttg1); changes in the location/activity of the ubiquitin-proteasome machinery via binding to valosin-containing protein (VCP/p97); and modified synaptic expression of proteins such as ubiquitin-activating enzyme E1 (Ube1).     

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum¹. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra². The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions^3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or βIII tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.     

Extracellular matrix: functions in the nervous system

An astonishing number of extracellular matrix glycoproteins are expressed in dynamic patterns in the developing and adult nervous system. Neural stem cells, neurons, and glia express receptors that mediate interactions with specific extracellular matrix molecules. Functional studies in vitro and genetic studies in mice have provided evidence that the extracellular matrix affects virtually all aspects of nervous system development and function. Here we will summarize recent findings that have shed light on the specific functions of defined extracellular matrix molecules on such diverse processes as neural stem cell differentiation, neuronal migration, the formation of axonal tracts, and the maturation and function of synapses in the peripheral and central nervous system.     

unc-44 Ankyrin and stn-2 gamma-syntrophin regulate sax-7 L1CAM function in maintaining neuronal positioning in Caenorhabditis elegans

The L1 family of single-pass transmembrane cell adhesion molecules (L1CAMs) is conserved from Caenorhabditis elegans and Drosophila to vertebrates and is required for axon guidance, neurite outgrowth, and maintenance of neuronal positions. The extracellular region of L1CAMs mediates cell adhesion via interactions with diverse cell-surface and extracellular matrix proteins. In contrast, less is known regarding the function of the intracellular domains in the L1CAM cytoplasmic tail. Previously, we identified a role of the C. elegans L1CAM homolog, SAX-7, in maintaining neuronal and axonal positioning. Here, we demonstrate that this function is dependent on three conserved motifs that reside in the SAX-7 cytoplasmic tail: (1) the FERM-binding motif, (2) the ankyrin-binding domain, and (3) the PDZ-binding motif. Furthermore, we provide molecular and genetic evidence that UNC-44 ankyrin and STN-2 γ-syntrophin bind SAX-7 via the respective ankyrin-binding and PDZ-binding motifs to regulate SAX-7 function in maintaining neuronal positioning.     

Sulfate and glucosamine labelling of the intercellular matrix in vitellogenic follicels of a moth

Summary InHyalophora cecropia the intercellular spaces of the follicles contain during vitellogenesis a matrix that can be labelled eitherin situ or in culture with³⁵S-sulfate and³H-glucosamine. The matrix was demonstrated by autoradiography and also by treating follicles for 15 min with pronase, which released TCA-soluble matrix fragments with molecular weights of up to 2×10⁶ daltons. Testicular hyaluronidase degraded the high molecular weight fragments, and thus it is probable that they are chondroitin sulfate-like mucopolysaccharides. With the termination of vitellogenesis new matrix is no longer deposited, and the pre-existing material is disassembled. The sulfated matrix may account for the patency of the intercellular diffusion channels essential for blood protein uptake and also for the low level, extracellular binding of blood proteins that characterizes the vitellogenic follicle inHyalophora.     

Extracellular Matrix in Neural Plasticity and Regeneration

The extracellular matrix (ECM) is a fundamental component of biological tissues. The ECM in the central nervous system (CNS) is unique in both composition and function. Functions such as learning, memory, synaptogenesis, and plasticity are regulated by numerous ECM molecules. The neural ECM acts as a non-specific physical barrier that modulates neuronal plasticity and axon regeneration. There are two specialized types of ECM in the CNS, diffuse perisynaptic ECM and condensed ECM, which selectively surround the perikaryon and initial part of dendritic trees in subtypes of neurons, forming perineuronal nets. This review presents the current knowledge about the role of important neuronal ECM molecules in maintaining the basic functions of a neuron, including electrogenesis and the ability to form neural circuits. The review mainly focuses on the role of ECM components that participate in the control of key events such as cell survival, axonal growth, and synaptic remodeling. Particular attention is drawn to the numerous molecular partners of the main ECM components. These regulatory molecules are integrated into the cell membrane or disposed into the matrix itself in solid or soluble form. The interaction of the main matrix components with molecular partners seems essential in molecular mechanisms controlling neuronal functions. Special attention is paid to the chondroitin sulfate proteoglycan 4, type 1 transmembrane protein, neural-glial antigen 2 (NG2/CSPG4), whose cleaved extracellular domain is such a molecular partner that it not only acts directly on neural and vascular cells, but also exerts its influence indirectly by binding to resident ECM molecules.

     

Molecular characterization of Pegarn: a <Emphasis Type="Italic">Drosophila</Emphasis> homolog of UNC-51 kinase

We have isolated and characterized the gene encoding a Drosophila melanogaster homolog of Caenorhabditis elegans UNC-51 (uncoordinated movement-51): Pegarn. Developmental Northern blot shows the Pegarn gene is expressed at all stages of development. The protein is detected throughout the Drosophila third instar larval central nervous system (CNS) in axons projecting out from the ventral ganglion and in the optic anlagen of the optic lobe. Heterozygous Pegarn mutant embryos show defects in larval axonal neuronal patterning, but survive to adulthood. Homozygous mutants have an even more deformed pattern of neuronal development and do not survive through the larval stages. The data from this research suggest the critical roles of Pegarn in CNS and PNS axonal formation in Drosophila melanogaster and indicates its similar role in other multicellular species.     

The contribution of β1 integrins to neuronal migration and differentiation depends on extracellular matrix molecules

The interaction of β1 integrin receptors and different extracellular matrix molecules during neuronal development was investigated by comparing both migration and morphological differentiation of D3 wild-type embryonic stem (ES) cell line-derived neural precursor cells with those of the β1 integrin knockout ES cell line G201. Analysing neurosphere explants on laminin and fibronectin as major β1 integrin ligands, the maximal spreading of outward migrating neuronal cells was determined. Compared with gelatine as a standard substrate, migration was found to be significantly increased for D3-derived neurospheres on fibronectin and laminin-1. These matrix effects were found to be even enhanced for G201 preparations. In addition, also the differentiation of wild-type and β1 integrin −/− neurones – as determined by MAP-2- and HNK-1-immunoreactive processes – was found to be increased on fibronectin and laminin when compared to gelatine standards. In the respective knockout preparations on these matrices, again perturbation effects were less pronounced than on gelatine. Our observations indicate that laminin and fibronectin are involved both in β1 integrin-dependent and -independent signalling mechanisms during neurogenesis. Upregulation of compensatory mechanisms such as β1 integrin-independent receptors for laminin and fibronectin might be responsible for the much less pronounced perturbations of G201 neural precursor migration and differentiation on these two substrates than on gelatine.     

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes ^{8, 16}. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades ¹⁴. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer''s, Huntington''s, and Multiple Sclerosis ^{4, 14, 3, 11, 7}.In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures ^{15, 5}, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) ^{15, 5}, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.^{6, 10}). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) ^{13, 12}. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis ⁹. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye ². The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time ^{1, 10}. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.     

Autophagy modulates cell migration and β1 integrin membrane recycling

Cell migration is dependent on a series of integrated cellular events including the membrane recycling of the extracellular matrix receptor integrins. In this paper, we investigate the role of autophagy in regulating cell migration. In a wound-healing assay, we observed that autophagy was reduced in cells at the leading edge than in cells located rearward. These differences in autophagy were correlated with the robustness of MTOR activity. The spatial difference in the accumulation of autophagic structures was not detected in rapamycin-treated cells, which had less migration capacity than untreated cells. In contrast, the knockdown of the autophagic protein ATG7 stimulated cell migration of HeLa cells. Accordingly, atg3^?/? and atg5^?/? MEFs have greater cell migration properties than their wild-type counterparts. Stimulation of autophagy increased the co-localization of β1 integrin-containing vesicles with LC3-stained autophagic vacuoles. Moreover, inhibition of autophagy slowed down the lysosomal degradation of internalized β1 integrins and promoted its membrane recycling. From these findings, we conclude that autophagy regulates cell migration, a central mechanism in cell development, angiogenesis, and tumor progression, by mitigating the cell surface expression of β1 integrins.     

Matrix metalloproteinase‐3 in the central nervous system: a look on the bright side

Matrix metalloproteinases (MMPs) are a large family of proteases involved in many cell‐matrix and cell‐cell signalling processes through activation, inactivation or release of extracellular matrix (ECM) and non‐ECM molecules, such as growth factors and receptors. Uncontrolled MMP activities underlie the pathophysiology of many disorders. Also matrix metalloproteinase‐3 (MMP‐3) or stromelysin‐1 contributes to several pathologies, such as cancer, asthma and rheumatoid arthritis, and has also been associated with neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and multiple sclerosis. However, based on defined MMP spatiotemporal expression patterns, the identification of novel candidate molecular targets and in vitro and in vivo studies, a beneficial role for MMPs in CNS physiology and recovery is emerging. The main purpose of this review is to shed light on the recently identified roles of MMP‐3 in normal brain development and in plasticity and regeneration after CNS injury and disease. As such, MMP‐3 is correlated with neuronal migration and neurite outgrowth and guidance in the developing CNS and contributes to synaptic plasticity and learning in the adult CNS. Moreover, a strict spatiotemporal MMP‐3 up‐regulation in the injured or diseased CNS might support remyelination and neuroprotection, as well as genesis and migration of stem cells in the damaged brain.     

Kir4.1-mediated spatial buffering of K+: Experimental challenges in determination of its temporal and quantitative contribution to K+ clearance in the brain

Neuronal activity results in release of K⁺ into the extracellular space of the central nervous system. If the excess K⁺ is allowed to accumulate, neuronal firing will be compromised by the ensuing neuronal membrane depolarization. The surrounding glial cells are involved in clearing K⁺ from the extracellular space by molecular mechanism(s), the identity of which have been a matter of controversy for over half a century. Kir4.1-mediated spatial buffering of K⁺ has been promoted as a major contributor to K⁺ removal although its quantitative and temporal contribution has remained undefined. We discuss the biophysical and experimental challenges regarding determination of the contribution of Kir4.1 to extracellular K⁺ management during neuronal activity. It is concluded that 1) the geometry of the experimental preparation is crucial for detection of Kir4.1-mediated spatial buffering and 2) Kir4.1 enacts spatial buffering of K⁺ during but not after neuronal activity.     

NudC is required for interkinetic nuclear migration and neuronal migration during neocortical development

NudC is a highly conserved protein necessary for cytoplasmic dynein-mediated nuclear migration in Aspergillus nidulans. NudC interacts genetically with Aspergillus NudF and physically with its mammalian orthologue Lis1, which is crucial for nuclear and neuronal migration during brain development. To test for related roles for NudC, we performed in utero electroporation into embryonic rat brain of cDNAs encoding shRNAs as well as wild-type and mutant forms of NudC. We show here that NudC, like Lis1, is required for neuronal migration during neocorticogenesis and we identify a specific role in apical nuclear migration in radial glial progenitor cells. These results identify a novel neuronal migration gene with a specific role in interkinetic nuclear migration, consistent with cytoplasmic dynein regulation.