Dominant-negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release.

Sympathetic neurons require nerve growth factor for survival and die by apoptosis in its absence. Key steps in the death pathway include c-Jun activation, mitochondrial cytochrome c release, and caspase activation. Here, we show that neurons rescued from NGF withdrawal-induced apoptosis by expression of dominant-negative c-Jun do not release cytochrome c from their mitochondria. Furthermore, we find that the mRNA for BIM(EL), a proapoptotic BCL-2 family member, increases in level after NGF withdrawal and that this is reduced by dominant-negative c-Jun. Finally, overexpression of BIM(EL) in neurons induces cytochrome c redistribution and apoptosis in the presence of NGF, and neurons injected with Bim antisense oligonucleotides or isolated from Bim(-/-) knockout mice die more slowly after NGF withdrawal.

c-Jun and the transcriptional control of neuronal apoptosis.

There has been considerable interest in the molecular mechanisms of apoptosis in mammalian neurons because this form of neuronal cell death is important for the normal development of the nervous system and because inappropriate neuronal apoptosis may contribute to the pathology of human neurodegenerative diseases. The aim of recent research has been to identify the key components of the cell death machinery in neurons and understand how the cell death programme is regulated by intracellular signalling pathways activated by the binding of neurotrophins or death factors to specific cell surface receptors. The aim of this commentary was to review research that has investigated the role of the Jun N-terminal kinase (JNK)/c-Jun signalling pathway in neuronal apoptosis, focusing in particular on work carried out with developing sympathetic neurons. Experiments with sympathetic neurons cultured in vitro, as well as with cerebellar granule neurons and differentiated PC12 cells, have demonstrated that JNK/c-Jun signalling can promote apoptosis following survival factor withdrawal. In addition, experiments with Jnk(-/-) knockout mice have provided evidence that Jnk3 may be required for apoptosis in the hippocampus in vivo following injection of kainic acid, an excitotoxin, and that Jnk1 and Jnk2 are required for apoptosis in the developing embryonic neural tube. However, in the embryonic forebrain, Jnk1 and Jnk2 have the opposite function and are necessary for the survival of developing cortical neurons. These results suggest that JNKs and c-Jun are important regulators of the cell death programme in the mammalian nervous system, but that their biological effects depend on the neuronal type and stage of development.

Blocking cytochrome c activity within intact neurons inhibits apoptosis.

Cytochrome c has been shown to play a role in cell-free models of apoptosis. During NGF withdrawal-induced apoptosis of intact rat superior cervical ganglion (SCG) neurons, we observe the redistribution of cytochrome c from the mitochondria to the cytoplasm. This redistribution is not inhibited by the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVADfmk) but is blocked by either of the neuronal survival agents 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (CPT-cAMP) or cycloheximide. Moreover, microinjection of SCG neurons with antibody to cytochrome c blocks NGF withdrawal-induced apoptosis. However, microinjection of SCG neurons with cytochrome c does not alter the rate of apoptosis in either the presence or absence of NGF. These data suggest that cytochrome c is an intrinsic but not limiting component of the neuronal apoptotic pathway.

CD44 exhibits a cell type dependent interaction with triton X-100 insoluble, lipid rich, plasma membrane domains.

CD44 is an abundant, widely expressed transmembrane glycoprotein which can act as a receptor for the extracellular matrix glycosaminoglycan, hyaluronan. Biochemical and morphological studies have demonstrated that in fibroblasts a significant of the CD44 population is resistant to Triton X-100 extraction and that the detergent insoluble protein is co-localized with components of the cortical cytoskeleton. Surprisingly, this distribution is not abrogated upon deletion of the CD44 cytoplasmic tail indicating that mechanisms other than a direct interaction with the cytoskeleton can regulate CD44. In this manuscript, the mechanisms underlying this detergent-insoluble association are further investigated. There was no evidence that the Triton X-100 insolubility of CD44 resulted from homotypic aggregation, an association with hyaluronan or from a direct, or indirect, association with the cytoskeleton. Instead, evidence is presented that the detergent insolubility of fibroblast CD44 at 4 degrees C results from an association of the CD44 transmembrane domain with Triton X-100 resistant, lipid rich, plasma membrane domains. The proportion of the CD44 found in these Triton X-100 insoluble structures is dependent upon cell type and cannot be altered by changing cell motility or extracellular matrix associations. These studies provide evidence for a novel mechanism regulating this adhesion protein in the plasma membrane.

Hyaluronan binding by CD44 is regulated by a phosphorylation-independent mechanism.

CD44 is an adhesion receptor for which the major characterized ligand is the extracellular matrix glycosaminoglycan, hyaluronan. This interaction underlies CD44-mediated cell attachment, cell migration, and matrix remodelling during development and wound healing. Truncation of the CD44 cytoplasmic domain does not prevent cell surface expression of this hyaluronan receptor but it dramatically impairs ligand binding. In this study we have examined the role of phosphorylation in regulating this function by mutating the target serine residues to either neutral amino acids with the aim of creating a phosphorylation-incompetent molecule, or to acidic residues to mimic a fully phosphorylated CD44. In transfected AKR1 cells the behavior of both the neutral and acidic mutants was indistinguishable from wild-type CD44, indicating that there is a phosphorylation-independent mechanism involved in regulating hyaluronan binding.

The cytoplasmic tail of CD44 is required for basolateral localization in epithelial MDCK cells but does not mediate association with the detergent-insoluble cytoskeleton of fibroblasts.

A number of recent reports on the trafficking of receptor proteins in MDCK epithelial cells have provided evidence that delivery to the basolateral domain requires a specific targeting sequence and that deletion of this sequence results in constitutive expression on the apical surface. To date, these studies have concentrated on receptors which are competent for internalization via the clathrin coated pits. We have examined the localization of a resident plasma membrane protein by transfecting human CD44 into MDCK cells. Using human specific and cross-species reactive antibodies, we show that in MDCK cells both the endogenous and transfected wild-type CD44 are found on the basolateral surface where they are restricted to the lateral domain. Deletion of the CD44 cytoplasmic tail reduces the half life of this mutant protein and causes it to be expressed both on the apical surface and to a significant extent within the cell. We have also used biochemical and morphological analysis to investigate the interaction of CD44 with the cytoskeleton in detergent extracted cells. Strikingly different extraction results were obtained between epithelial and fibroblast cells. However, there is no difference in the Triton X-100 solubility of the transfected wild-type and tail-less CD44 in fibroblasts and both forms of the protein remain associated with the cortical cytoskeleton after Triton X-100 extraction. These results demonstrate that the sequence present in the cytoplasmic domain of CD44 responsible for its distribution in epithelial cells is functionally and spatially separate from the ability of this protein to associate with the cytoskeleton.

Phosphorylation of CD44 in vivo requires both Ser323 and Ser325, but does not regulate membrane localization or cytoskeletal interaction in epithelial cells.

CD44 has been implicated to play an important role in a diverse range of physiological processes, which involve cell-matrix recognition, cell-cell adhesion and cell motility. There is increasing evidence that the highly conserved intracellular domain of CD44 may be involved in influencing these activities. CD44 is phosphorylated in vivo on serine residue(s). In view of the importance that phosphorylation has been accorded in a multitude of cellular regulatory processes, we have investigated the role of phosphorylation in the control of CD44. In this report we identify the sites of human CD44 phosphorylation by mutating the three conserved cytoplasmic serine residues. We show that both Ser323 and Ser325, but not Ser316, are required for phosphorylation in vivo and demonstrate that this event is not stimulated by phorbol esters. Clonal MDCK cell lines expressing both the single and double CD44 phosphorylation mutants have been generated. These cell lines have been used to directly assess the role of phosphorylation on CD44 localization in polarized epithelial cells and its association with the cytoskeleton.

A comparison of the effect of several factors on the plasminogen activator activity of cloned lines from an ethylnitrosourea-induced glioma and from normal tissue.

Cells from gliomas induced by N-ethyl-N-nitrosourea have a high basal level of plasminogen activator activity compared with cells from normal tissue. Plasminogen activator activity is known to be affected by many substances but whether inhibition or stimulation occurs depends on the cell and agent involved. It is not clear whether tumour and control cells from the same type of tissue respond similarly. A comparison has been made of the effect of several factors on both cell associated and secreted enzyme activity of cloned lines from a glioma and normal tissue. The effect of two cAMP elevating compounds was stimulatory while that of the steroid, dexamethasone, was generally inhibitory for both cells. However, the polypeptide hormone, epidermal growth factor, had a differential effect. It caused an increase in secreted enzyme activity in the tumour line but had no such effect on the control clone. The precise mechanism by which this occurs is unknown. Co-operative effects of the enzyme and growth hormone could result in more aggressive behaviour of the tumour cells.

Microcomputer system for the analysis of muscle function.

Though skeletal muscle function has recently been increasingly studied, a system for routine clinical examination in patients is not widely available. A microcomputer based system has been developed for the measurement and analysis of force-frequency characteristics, relaxation rate, fatiguability and endurance in the adductor pollicis muscle. The mobile system consists of a modified displacement transducer fitted with a linear-output Hall-effect sensor, a specially designed signal processing unit and a microcomputer for control of both the electrical stimulus and a suite of menu-driven, user-interactive programs. One hundred normal volunteers have been studied to produce a normal database for comparison with malnourished surgical patients. The system compares favourably with other systems in use for objective measurement of skeletal muscle function and introduces a refined technique for evaluating endurance.