Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton.

Neurofilaments in the axons of mammalian spinal cord neurons are extensively cross-linked; consequently, the filaments and their cross-bridges compose a three-dimensional lattice. We have used antibody decoration in situ combined with tissue preparation by the quick-freeze, deep-etch technique to locate three neurofilament polypeptides (195, 145, and 73 Kd) within this lattice. When antibodies against each polypeptide were incubated with detergent-extracted, formaldehyde-fixed samples of rabbit spinal cord, each antibody assumed a characteristic distribution: anti-73-Kd decorated the neurofilament core uniformly, but not the cross-bridges; anti-145-Kd also decorated the core, but less uniformly; sometimes the anti-145-Kd antibodies were located over the bases of cross-bridges. In contrast, anti-195-Kd primarily decorated the cross-bridges between the neurofilaments. These observations show that the 73-Kd polypeptide is a component of the central core of neurofilaments, and that the 195-Kd polypeptide is a component of the inter-neurofilamentous cross-bridges. It is consistent with this conclusion that we found few cross-bridges between neurofilaments in the optic nerves of neonatal rabbits during a developmental period when the ratio of 195 to 73 or 145-Kd polypeptides is much lower than in adults. The ratio of 195-Kd polypeptide to the other two neurofilament polypeptides also appeared much lower in the cell bodies and dendrites than in axons of adult spinal cord neurons, when the dispositions of the three polypeptides were studied by immunofluorescence experiments. The cell bodies apparently contain neurofilaments composed primarily of 145- and 73-Kd polypeptides, because we observed antibody decoration of individual neurofilaments in the cell bodies with anti-73- and -145-Kd, but not with anti-195-Kd. We conclude that the 195-Kd polypeptide participates in a cross-linking function, and that this function is, at least in certain neurons, most prevalent in the mature axon.

Identification of small molecule inhibitors of beta-amyloid cytotoxicity through a cell-based high-throughput screening platform.

Calpain activation is hypothesized to be an early occurrence in the sequence of events resulting in neurodegeneration, as well as in the signaling pathways linking extracellular accumulation of beta-amyloid (Abeta) peptides and intracellular formation of neurofibrillary tangles. In an effort to identify small molecules that prevent neurodegeneration in Alzheimer's disease by early intervention in the cell death cascade, a cell-based assay in differentiated Sh-SY5Y cells was developed using calpain activity as a read-out for the early stages of death in cells exposed to extracellular Abeta. This assay was optimized for high-throughput screening, and a library of approximately 120,000 compounds was tested. It was expected that the compounds identified as calpain inhibitors would include those that act directly on the enzyme and those that prevented calpain activation by blocking an upstream step in the pathway. In fact, of the compounds that inhibited calpain activation by Abeta with IC(50) values of <10 microM and showed little or no toxicity at concentrations up to 30 microM, none inhibit the calpain enzyme directly.

Neurotrophic 3,9-bis[(alkylthio)methyl]-and-bis(alkoxymethyl)-K-252a derivatives.

A series of 3,9 disubstituted [(alkylthio)methyl]- and (alkoxymethyl)-K-252a derivatives was synthesized with the aim of enhancing and separating the neurotrophic properties from the undesirable NGF (trk A kinase) and PKC inhibitory activities of K-252a. Data from this series reveal that substitution in the 3- and 9-positions of K-252a with these groups reduces trk A kinase inhibitory properties approximately 100- to > 500-fold while maintaining or in certain cases enhancing the neurotrophic activity. From this research, 3,9-bis[(ethylthio)methyl]-K-252a (8) was identified as a potent and selective neurotrophic agent in vitro as measured by enhancement of choline acetyltransferase activity in embryonic rat spinal cord and basal forebrain cultures. Compound 8 was found to have weak kinase inhibitory activity for trk A, protein kinase C1 protein kinase A, and myosin light chain kinase. On the basis of the in vitro profile, 8 was evaluated in in vivo models suggestive of neurological diseases. Compound 8 was active in preventing degeneration of cholinergic neurons of the nucleus basalis magnocellularis (NBM) and reduced developmentally programmed cell death (PCD) of female rat spinal nucleus of the bulbocavernosus motoneurons and embryonic chick lumbar motoneurons.

CEP-1347/KT7515, a JNK pathway inhibitor, supports the in vitro survival of chick embryonic neurons.

Developing neurons depend on target-derived trophic factors for survival in vivo and in vitro, which also decrease the activity of c-Jun N-terminal kinase (JNK). We have recently described a survival-promoting effect of inhibitors of cyclin-dependent kinases and JNK on chick peripheral embryonic neurons. Here, we report that the small trophic molecule CEP-1347/KT7515, which has been shown to inhibit the JNK signalling pathway, can promote long term-survival of cultured chick embryonic dorsal root ganglion, sympathetic, ciliary and motor neurons. Because of their pharmacological properties, small trophic molecules such as CEP-1347/KT7515 might be of interest for the treatment of neurodegenerative disorders.

Localization of motoneurons controlling the extraocular muscles of the rat.

The localization of extraocular motoneurons in the rat was investigated by injecting horseradish peroxidase and [125I]wheat germ agglutinin as retrograde tracer substances into individual eye muscles. The organization of subnuclei was found to be most similar to the rabbit. The subgroups representing the medial rectus and inferior rectus muscles are located in the rostral two thirds of the ipsilateral oculomotor nucleus (nIII) with some medial rectus motoneurons scattered laterally along the edge of the medial longitudinal fasciculus. The motor pool controlling the inferior oblique muscle is located in the middle third of the ipsilateral nIII. The motoneurons of the superior rectus muscle are in the caudal two-thirds of contralateral nIII while the levator palpebrae muscle has a bilateral innervation in the oculomotor nucleus. The motoneurons of the superior oblique are located in the contralateral trochlear nucleus although a few labeled neurons were scattered laterally in amongst the fibers of the medial longitudinal fasciculus. The cell bodies of lateral rectus motoneurons are not homogeneously distributed throughout the ipsilateral abducens nucleus. A regional separation between the latter and internuclear neurons was found after injecting HRP into the oculomotor nucleus.

Use of the B-IIb tetanus toxin derived fragment as a specific neuropharmacological transport agent.

It has been previously demonstrated that the non-toxic B-IIb tetanus toxin-derived fragment is transported retrogradely within neurons of the rat peripheral nervous system. In the present work we have shown that when a foreign polypeptide, in this case the Ibc tetanus toxin fragment, is coupled to B-IIb by a disulfide bond, it is transported retrogradely from the axonal endings within muscle to the motoneuronal perikarya. In contrast, Ibc fragment alone was found not to be transported. From these results we draw the conclusion that fragments like B-IIb may serve as specific carriers for chemical and chemotherapeutic agents into the central nervous system.

A loss of FUS/TLS function leads to impaired cellular proliferation.

Fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) is a multifunctional RNA/DNA-binding protein that is pathologically associated with cancer and neurodegeneration. To gain insight into the vital functions of FUS and how a loss of FUS function impacts cellular homeostasis, FUS expression was reduced in different cellular models through RNA interference. Our results show that a loss of FUS expression severely impairs cellular proliferation and leads to an increase in phosphorylated histone H3, a marker of mitotic arrest. A quantitative proteomics analysis performed on cells undergoing various degrees of FUS knockdown revealed protein expression changes for known RNA targets of FUS, consistent with a loss of FUS function with respect to RNA processing. Proteins that changed in expression as a function of FUS knockdown were associated with multiple processes, some of which influence cell proliferation including cell cycle regulation, cytoskeletal organization, oxidative stress and energy homeostasis. FUS knockdown also correlated with increased expression of the closely related protein EWS (Ewing's sarcoma). We demonstrate that the maladaptive phenotype resulting from FUS knockdown is reversible and can be rescued by re-expression of FUS or partially rescued by the small-molecule rolipram. These results provide insight into the pathways and processes that are regulated by FUS, as well as the cellular consequences for a loss of FUS function.

Persistent ectopic expression of Drosophila homeotic genes resulting from maternal deficiency of the extra sex combs gene product.

Like other members of the Polycomb group, the extra sex combs gene (esc) is required for the correct repression of loci in the major homeotic gene complexes. We show here that embryos lacking both maternal and zygotic esc+ function display transient, general derepression of both the Ultrabithorax (Ubx) and Antennapedia (Antp) genes during germ band shortening, but Sex combs reduced (Scr) expression is almost normal in the epidermis and lacking in the central nervous system (CNS). In addition, embryos that are maternally esc- but receive two paternal copies of esc+ often are characterized by ectopic expression of the three homeotic genes, especially Ubx and Antp in the CNS. Imaginal discs from these paternally rescued embryos may show discrete patches of expression of Ubx and Scr in inappropriate locations. Thus, lack of esc+ function during a brief period in early embryogenesis results in a heritable change in determined state, even in a genetically wild type animal. Within these ectopic patches, homeotic gene expression may be regulated by the disc positional fields and by cross-regulatory interactions between homeotic genes.

Misregulation of homeotic gene expression in Drosophila larvae resulting from mutations at the extra sex combs locus.

Using monoclonal antibodies specific for their protein products, the expression of the Ubx, Antp, and Scr genes was examined in imaginal discs and central nervous systems of esc-Drosophila larvae. In esc-mutants, both the Ubx and Scr proteins are expressed at increased levels or in new locations in the leg discs. Ubx also is expressed in new locations in the posterior wing disc and in small groups of cells in the antenna disc. The Antp protein is expressed ectopically in the eye-antenna disc; however, obvious abnormal expression of Antp was not found in the thoracic imaginal discs. Particularly striking is the fact that a single disc, such as the mesothoracic leg, can show increased expression of both a more "anterior" homeotic gene (Scr) and a more "posterior" gene (Ubx). Ectopic expression of Ubx and Antp, but not of Scr, is seen in the central nervous system of mutant larvae. These results are discussed with respect to the adult esc-phenotype and the differential effects of esc mutations on early and late development.

Expression of the Sex combs reduced protein in Drosophila larvae.

We have generated a monoclonal antibody that binds specifically to the protein product of the homeotic Sex combs reduced (Scr) gene of Drosophila, and have mapped the patterns of Scr expression in late third instar larvae. Virtually the entire prothoracic leg imaginal disc expresses the gene, although the levels of expression vary in different disc regions. This heterogeneity does not reflect the compartmental domains defined by engrailed gene expression. Expression is also observed in the cells of the humeral and labial discs, and there is a small patch of Scr-expressing cells in the antenna disc. The gene is expressed in adepithelial cells of the three thoracic leg discs, but not in the wing or haltere discs. In the central nervous system, Scr expression is confined to a narrow band of cells in the subesophageal region of the ventral ganglion. The results are discussed with respect to the known genetic requirements for Scr+ function.

Separation of neurofilaments from ATPase activity by precipitation with anti-neurofilament antibodies.

The possibility that neurofilaments could be involved in the transduction of chemical and mechanical energy in axons led us to investigate whether neurofilament proteins can hydrolyze ATP. We fractionated neurofilaments from rabbit spinal cord and found that preparations highly enriched for neurofilament proteins hydrolyzed ATP at a substantial rate (as high as 0.4 mumol/min/mg protein). However, the ATPase activity was neither inhibited by anti-neurofilament antibody, nor was it precipitated by the antibody under circumstances that precipitated most of the neurofilament polypeptides. We conclude that neurofilament proteins do not hydrolyze ATP at a significant rate under the conditions of our assay; if hydrolysis of ATP is a physiological function of neurofilaments, additional factors are required.

Differentiation of motor nerve terminals formed in the absence of muscle fibres.

During reinnervation of frog skeletal muscle, axons form functional nerve terminals at original synaptic sites on denervated myofibres. When muscle is damaged as well as denervated, myofibres decompose but their sheaths of basal lamina (BL) survive. Despite the absence of myofibres, axons regenerate to contact BL and there acquire clusters of synaptic vesicles and membrane-associated dense patches that resemble active zones; BL regulates this differentiation. We show here that these BL-associated axonal segments appear smaller and contain fewer active zones than terminals on intact myofibres in the same preparation. However, terminals formed on BL sheaths are capable of activity-dependent recycling of synaptic vesicles (demonstrated by tracer uptake), and bear an antigen normally present in terminals but not preterminal axons (demonstrated by immunofluorescence). Thus, axons can acquire functional and biochemical, as well as morphological, characteristics of normal motor nerve terminals in the absence of a postsynaptic cell.

Posttranslational modification of neurofilament polypeptides in rabbit retina.

Three polypeptides that compose neurofilaments, designated H, M, and L, are synthesized in the cell bodies of neurons and subsequently conveyed down their axons by the process of slow axonal transport. The axonal form of H, which is a component of the cross bridges between the neurofilaments, is antigenically different from the form in the cell bodies and dendrites. To understand how this special form of H is directed to the axon, and more generally how intracellular differentiation is established and maintained by the selective delivery of different molecular species to different compartments of a cell, we have studied the events that occur immediately after the synthesis of the three neurofilament polypeptides in the retinas of rabbits. We observed that H and M are synthesized in the retina as precursor polypeptides, EH and EM, that migrate markedly faster on SDS polyacrylamide gels than their mature axonal forms. The maturation of these precursors requires more than one day and appears to involve their phosphorylation. Only the electrophoretically mature forms appear in the axons of the retinal ganglion cells in the optic nerve. We consider the following interpretation of these observations. Shortly after they are translated in the cell body, the neurofilament polypeptides become phosphorylated at multiple sites. However, only after they have moved a distance of several hundred micrometers down the axon, H and M are phosphorylated at additional sites, causing their conformation or binding properties to change. This change, which is reflected in the reduction of their electrophoretic mobility and the appearance of new antigenic determinants, may function to alter the H-mediated crossbridges and produces the morphological and structural properties of the neurofilament lattice that is characteristic of axons.

Potential utility of rhIGF-1 in neuromuscular and/or degenerative disease.

Neuromuscular/neurodegenerative disorders, such as the death of spinal cord motor neurons in amyotrophic lateral sclerosis (ALS) or the degeneration of spinal cord motor neuron axons in certain peripheral neuropathies, present a unique opportunity for therapeutic intervention with neurotrophic proteins. We have found that in mixed rat embryonic spinal cord cultures or in purified motor neuron preparations, recombinant human insulin-like growth factor 1 (rhIGF-1) enhances the survival of motor neurons at EC50 concentrations of 2 nM, consistent with an interaction at the tyrosine kinase-coupled rhIGF-1 receptor. In a model of programmed cell death in ovo, administration of rhIGF-1 produces a marked survival of motor neurons. In a variety of models of predominantly motor neuron or nerve injury in rodents, administration of rhIGF-1 prevents the death of motor neurons in neonatal facial nerve lesions, attenuates the loss of cholinergic phenotype in adult hypoglossal nerve axotomy and hastens recovery from sciatic nerve crush in mice. In a genetic model of motor neuron compromise, the wobbler mouse, rhIGF-1 (1 mg/kg s.c. daily) delayed the deterioration of grip strength and provided for a more normal distribution of fibre types. In addition, rhIGF-1 (0.3-1.0 mg/kg s.c. daily) prevents the motor and/or sensory neuropathy in rodents caused by vincristine, cisplatinum or Taxol. These combined data indicate that rhIGF-1 has marked effects on the survival of compromised motor neurons and the maintenance of their axons and functional connections. They also suggest the potential utility of rhIGF-1 for the treatment of diseases such as ALS and certain neuropathies.

K-252a promotes survival and choline acetyltransferase activity in striatal and basal forebrain neuronal cultures.

The organic molecule K-252a promoted cell survival, neurite outgrowth, and increased choline acetyltransferase (ChAT) activity in rat embryonic striatal and basal forebrain cultures in a concentration-dependent manner. A two- to threefold increase in survival was observed at 75 nM K-252a in both systems. A single application of K-252a at culture initiation prevented substantial (> 60%) cell death that otherwise occurred after 4 days in striatal or basal forebrain cultures. A 5-h exposure of striatal or basal forebrain cells to K-252a, followed by its removal, resulted in survival equivalent to that observed in cultures continually maintained in its presence. This is in contrast to results found with a 5-h exposure of basal forebrain cultures to nerve growth factor (NGF). Acute exposure of basal forebrain cultures to K-252a, but not to NGF, increased ChAT activity, indicating that NGF was required the entire culture period for maximum activity. Striatal cholinergic and GABAergic neurons were among the neurons rescued by K-252a. Of the protein growth factors tested in striatal cultures (ciliary neurotrophic factor, neurotrophin-3, NGF, brain-derived neurotrophic factor, interleukin-2, basic fibroblast growth factor), only brain-derived neurotrophic factor promoted survival. The enhancement of survival and ChAT activity of basal forebrain and striatal neurons by K-252a defines additional populations of neurons in which survival and/or differentiation is regulated by a K-252a-responsive mechanism. The above results expand the potential therapeutic targets for these molecules for the treatment of neuro-degenerative diseases.

Segmentally repeated pattern of expression of a cell surface glycoprotein in Drosophila embryos.

We report here that a previously described cell surface antigen (Brower, Smith & Wilcox, 1980) is expressed in a segmentally repeating pattern of stripes in the epidermis and nervous system of segmented Drosophila embryos. We also report that the antigenic activity is found on two closely related cell surface glycoproteins. The pattern of expression of this antigen is reminiscent of the expression of some segmentation genes and is affected by mutation of at least two of these genes, fushi tarazu and paired. Thus these glycoproteins are candidates for cell surface molecules involved in carrying out the patterning processes controlled by segmentation genes.

K-252a induces tyrosine phosphorylation of the focal adhesion kinase and neurite outgrowth in human neuroblastoma SH-SY5Y cells.

The protein kinase inhibitor K-252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chick dorsal root ganglion cultures. To determine the mechanism by which K-252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH-SY5Y. K-252a induced neurite outgrowth in a dose-dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125- and 140-kDa proteins. The phosphorylation events were independent of protein kinase C inhibition because down-regulation of protein kinase C by long-term treatment with phorbol ester did not prevent K252a-induced tyrosine phosphorylation. Similarly, the protein kinase C inhibitors H7, GF-109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine kinase (Fak). Induction of phosphorylation coincided with increased Fak activity and appeared to be independent of ligand/integrin interaction. The induction of Fak phosphorylation by K-252a was also observed in LA-N-5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein kinase C-independent induction of tyrosine phosphorylation and the identification of Fak as a substrate of K-252a-induced tyrosine kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.