Friedrich Propst

Culture and characteristics of hormone-responsive neuroblastoma X glioma hybrid cells.

Publisher Summary Due to the complexity of the mammalian nervous system, results from biochemical or pharmacological experiments with pieces or homogenates of nervous tissue are difficult to interpret. Only recently, a few cases have been reported of apparently relatively homogeneous populations of certain cell types from nervous tissue. This chapter studies molecular mechanisms of nervous tissue functions that have been resting completely on model systems derived from tumors of the nervous system and describes the methodology for handling tumor cell lines of neuronal character. By somatic cell hybridization, several hybrid cell lines have been generated from mouse neuroblastoma and rat glioma cells, some of which display a long list of neuronal properties and in which these differentiated functions are well expressed. Therefore, these cells are especially suited for studying such functions among which are susceptibilities to peptide hormones.

Microtubule-associated protein 1B controls directionality of growth cone migration and axonal branching in regeneration of adult dorsal root ganglia neurons.

During development, microtubule-associated protein 1B (MAP1B) is one of the earliest MAPs, preferentially localized in axons and growth cones, and plays a role in axonal outgrowth. Although generally downregulated in the adult, we have shown that MAP1B is constitutively highly expressed in adult dorsal root ganglia (DRGs) and associated with central sprouting and peripheral regeneration of these neurons. Mutant mice with a complete MAP1B null allele that survive until adulthood exhibit a reduced myelin sheath diameter and conductance velocity of peripheral axons and lack of the corpus callosum. Here, to determine the function of MAP1B in axonal regeneration, we used cultures of adult DRG explants and/or dissociated neurons derived from this map1b-/- mouse line. Whereas the overall length of regenerating neurites lacking MAP1B was similar to wild-type controls, our analysis revealed two main defects. First, map1b-/- neurites exhibited significantly (twofold) higher terminal and collateral branching. Second, the turning capacity of growth cones (i.e., “choice” of a proper orientation) was impaired. In addition, lack of MAP1B may affect the post-translational modification of tubulin polymers: quantitative analysis showed a reduced amount of acetylated microtubules within growth cones, whereas the distribution of tyrosinated or detyrosinated microtubules was normal. Both growth cone turning and axonal branch formation are known to involve local regulation of the microtubule network. Our results demonstrate that MAP1B plays a role in these processes during plastic changes in the adult. In particular, the data suggest MAP1B implication in the locally coordinated assembly of cytoskeletal components required for branching and straight directional axon growth.

Distinct Roles of c-Jun N-Terminal Kinase Isoforms in Neurite Initiation and Elongation during Axonal Regeneration

c-Jun N-terminal kinases (JNKs) (comprising JNK1–3 isoforms) are members of the MAPK (mitogen-activated protein kinase) family, activated in response to various stimuli including growth factors and inflammatory cytokines. Their activation is facilitated by scaffold proteins, notably JNK-interacting protein-1 (JIP1). Originally considered to be mediators of neuronal degeneration in response to stress and injury, recent studies support a role of JNKs in early stages of neurite outgrowth, including adult axonal regeneration. However, the function of individual JNK isoforms, and their potential effector molecules, remained unknown. Here, we analyzed the role of JNK signaling during axonal regeneration from adult mouse dorsal root ganglion (DRG) neurons, combining pharmacological JNK inhibition and mice deficient for each JNK isoform and for JIP1. We demonstrate that neuritogenesis is delayed by lack of JNK2 and JNK3, but not JNK1. JNK signaling is further required for sustained neurite elongation, as pharmacological JNK inhibition resulted in massive neurite retraction. This function relies on JNK1 and JNK2. Neurite regeneration of jip1−/− DRG neurons is affected at both initiation and extension stages. Interestingly, activated JNKs (phospho-JNKs), as well as JIP1, are also present in the cytoplasm of sprouting or regenerating axons, suggesting a local action on cytoskeleton proteins. Indeed, we have shown that JNK1 and JNK2 regulate the phosphorylation state of microtubule-associated protein MAP1B, whose role in axonal regeneration was previously characterized. Moreover, lack of MAP1B prevents neurite retraction induced by JNK inhibition. Thus, signaling by individual JNKs is differentially implicated in the reorganization of the cytoskeleton, and neurite regeneration.

Mapmodulin/Leucine-rich Acidic Nuclear Protein Binds the Light Chain of Microtubule-associated Protein 1B and Modulates Neuritogenesis

We had previously described the leucine-rich acidic nuclear protein (LANP) as a candidate mediator of toxicity in the polyglutamine disease, spinocerebellar ataxia type 1 (SCA1). This was based on the observation that LANP binds ataxin-1, the protein involved in this disease, in a glutamine repeat-dependent manner. Furthermore, LANP is expressed abundantly in purkinje cells, the primary site of ataxin-1 pathology. Here we focused our efforts on understanding the neuronal properties of LANP. In undifferentiated neuronal cells LANP is predominantly a nuclear protein, requiring a bona fide nuclear localization signal to be imported into the nucleus. LANP translocates from the nucleus to the cytoplasm during the process of neuritogenesis, interacts with the light chain of the microtubule-associated protein 1B (MAP1B), and modulates the effects of MAP1B on neurite extension. LANP thus could play a key role in neuronal development and/or neurodegeneration by its interactions with microtubule associated proteins.

S-nitrosylation of microtubule-associated protein 1B mediates nitric-oxide-induced axon retraction

Treatment of cultured vertebrate neurons with nitric oxide leads to growth-cone collapse, axon retraction and the reconfiguration of axonal microtubules. We show that the light chain of microtubule-associated protein (MAP) 1B is a substrate for S-nitrosylation in vivo, in cultured cells and in vitro. S-nitrosylation occurs at Cys 2457 in the COOH terminus. Nitrosylation of MAP1B leads to enhanced interaction with microtubules and correlates with the inhibition of neuroblastoma cell differentiation. We further show, in dorsal root ganglion neurons, that MAP1B is necessary for neuronal nitric oxide synthase control of growth-cone size, growth-cone collapse and axon retraction. These results reveal an S-nitrosylation-dependent signal-transduction pathway that is involved in regulation of the axonal cytoskeleton and identify MAP1B as a major component of this pathway. We propose that MAP1B acts by inhibiting a microtubule- and dynein-based mechanism that normally prevents axon retraction.

MAP1B coordinates microtubule and actin filament remodeling in adult mouse Schwann cell tips and DRG neuron growth cones

We previously described the function of MAP1B in both turning and branching of regenerating neurites. Our results suggested implication of MAP1B in coupling of actin and microtubule movements, a hypothesis investigated here using DRG neurons and Schwann cells (SCs), which also transiently express MAP1B. Cell motility and cytoskeletal rearrangements were assessed before and after addition of lysophosphatidic acid (LPA), an extracellular signaling phospholipid triggering changes in actin distribution and cell morphology. First, we show that MAP1B is required for SC migration in vitro, extending our previous work on its function in growth cone motility. Second, LPA stimulation induces drastic retraction of processes from MAP1B-expressing cells in a two-step process: actin contraction, which is followed by microtubule backfolding. More importantly, we provide evidence that MAP1B is required for microtubule backfolding, thereby unravelling an important molecular mechanism implicated in coupling the movements of actin and microtubules during process retraction of neural cells.

Opioids, Noradrenaline and GTP Analogs Inhibit Cholera Toxin Activated Adenylate Cyclase in Neuroblastoma × Glioma Hybrid Cells

Abstract: D‐Ala2‐Met5‐enkephalin, morphine, and noradrenaline inhibit the adenylate cyclase in homogenates of neuroblastoma × glioma hybrid cells in a dose‐dependent manner even after the enzyme has been preactivated by cholera toxin. Half‐maximal inhibition and extent of inhibition are the same with native or cholera toxin‐activated enzyme. The inhibitions caused by opioids or noradrenaline are antagonized by naloxone or phentolamine, respectively. The effect of D‐Ala2‐Met5‐enkephalin on cholera toxin‐activated enzyme is immediate in onset and rapidly reversed by the addition of naloxone. Guanyl‐5′‐yl‐imidodiphosphate stimulates basal activity but inhibits the enzyme activated by cholera toxin or prostaglandin E1. Stimulation occurs at a concentration of 100 μM or above, inhibition even at 0.1 μM. The inhibitory effect of the non‐hydrolysable GTP analog is antagonized by GTP. Guanyl‐5′‐yl‐methylenediphosphonate, another nonhydrolysable GTP analog, inhibits basal as well as cholera toxin‐stimulated or prostaglandin E1‐stimulated adenylate cyclase. Other guanine derivatives such as GDP, GMP, cyclic GMP, guanyl‐5′‐yl‐phosphoric acid amide and guanosine have no effect under the same conditions. The results may be taken as a piece of evidence for two separate guanyl nucleotide‐binding sites accompanying the adenylate cyclase in the hybrid cells and mediating, respectively, stimulation and inhibition of the enzyme by hormones.

Targeted Inactivation of a Developmentally Regulated Neural Plectin Isoform (Plectin 1c) in Mice Leads to Reduced Motor Nerve Conduction Velocity

Cytolinker proteins stabilize cells mechanically, regulate cytoskeleton dynamics, and provide scaffolds for signaling molecules. For plectin, the prototype of these proteins, an unusual diversity of isoforms has been reported, which show distinct expression patterns, subcellular localizations, and functions. Plectin has been shown to have important functions in skin and muscle, but little is known about its role in neural cells. To address this issue, we generated two knock-out mouse lines, one which was selectively lacking plectin 1c (P1c), the major isoform expressed in neural cells, and another in which plectin was conditionally deleted in neuronal precursor cells. Using isoform-specific antibodies, we found P1c to be expressed late in development and to associate with postsynaptic dendrites of central nervous system neurons, motorneurons of spinal cord, sciatic nerve axons, and Schwann cells. Motor nerve conduction velocity was found significantly reduced in sciatic nerve from P1c-deficient as well as from conditional knock-out mice. This defect was traceable to an increased number of motor nerve fibers with small cross-sectional areas; the thicknesses of axons and of myelin sheaths were unaffected. This is the first report demonstrating an important role of plectin in a major nerve function.

The GSK3–MAP1B pathway controls neurite branching and microtubule dynamics

The microtubule-associated protein MAP1B plays a key role in axon regeneration. We investigated the role of GSK3-mediated MAP1B phosphorylation in local fine-tuning of neurite branching and the underlying microtubule (MT) dynamics. In wildtype adult dorsal root ganglia (DRG) neurons, MAP1B phosphorylation is locally reduced at branching points, and branching dynamics from growth cones and distal neurite shafts is increased upon GSK3 inhibition. While map1b-/- neurites, that display increased branching, are not affected by GSK3 inhibition, transfection of map1b-/- neurons with full-length map1b-cDNA restores the wildtype branching phenotype, demonstrating that MAP1B is a key effector downstream of GSK3. Experiments in mutant mice lacking tyrosinated MTs indicate a preferential association of phospho-MAP1B with tyrosinated MTs. Interestingly, inhibition of GSK3-mediated MAP1B phosphorylation in map1b-cDNA-transfected fibroblasts protects both tyrosinated and acetylated MTs from nocodazole-induced depolymerization, while detyrosinated MTs are less abundant in the presence of MAP1B. Our data thus provide new insight into the molecular link between GSK3, MAP1B, neurite branching and MT stability regulation. We suggest that, at branching points, MAP1B undergoes a fine regulation of both its phosphorylation and sub-cellular amounts, in order to modulate the local balance between acetylated, detyrosinated, and tyrosinated microtubule pools.

A 45 amino acid residue domain necessary and sufficient for proteolytic cleavage of the MAP1B polyprotein precursor

The microtubule‐associated proteins 1B and 1A are synthesized as polyprotein precursors which are rapidly cleaved to give rise to heavy and light chains constituting the respective microtubule‐associated protein 1B or microtubule‐associated protein 1A complex. To identify domains necessary for precursor processing, we expressed microtubule‐associated protein 1B deletion mutants in fibroblasts and monitored proteolytic cleavage of the precursor proteins by immunoblot analysis. We found that a novel hydrophilic, proline‐rich 45 amino acid domain containing the cleavage site is necessary and sufficient for processing. This domain is conserved in microtubule‐associated protein 1A. Additional sequences in the N‐terminal half of the heavy chain contribute to the efficiency of cleavage.