NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration

Neurodegeneration can be triggered by genetic or environmental factors. Although the precise cause is often unknown, many neurodegenerative diseases share common features such as protein aggregation and age dependence. Recent studies in Drosophila have uncovered protective effects of NAD synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) against activity-induced neurodegeneration and injury-induced axonal degeneration. Here we show that NMNAT overexpression can also protect against spinocerebellar ataxia 1 (SCA1)-induced neurodegeneration, suggesting a general neuroprotective function of NMNAT. It protects against neurodegeneration partly through a proteasome-mediated pathway in a manner similar to heat-shock protein 70 (Hsp70). NMNAT displays chaperone function both in biochemical assays and cultured cells, and it shares significant structural similarity with known chaperones. Furthermore, it is upregulated in the brain upon overexpression of poly-glutamine expanded protein and recruited with the chaperone Hsp70 into protein aggregates. Our results implicate NMNAT as a stress-response protein that acts as a chaperone for neuronal maintenance and protection. Our studies provide an entry point for understanding how normal neurons maintain activity, and offer clues for the common mechanisms underlying different neurodegenerative conditions.     

Expression of a mammalian Na-H exchanger in muscle fibres of the giant barnacle

It is now well established that the internal pH (pHi) of mammalian cells is regulated by means of a plasma membrane transport system that exchanges extracellular Na+ for intracellular H+ (ref. 1). Furthermore, modulation of the activity of the Na-H exchanger seems to have a crucial role in the action of various mitogens and growth factors. The possibility that such a mammalian Na-H exchanger might be efficiently expressed in a giant invertebrate cell was suggested to us by recent results of Barnard and Miledi and colleagues, who demonstrated in frog oocytes the expression of various plasma membrane channels that presumably were encoded by the mammalian messenger RNA wih which the oocytes had been injected. We used muscle fibres of the giant barnacle, which normally have no demonstrable Na-H exchanger activity, and report here that, when injected with poly(A)+ RNA isolated from rabbit liver, the muscle fibres express a Na-H exchanger. No such expression is observed, however, when the injected material is pretreated with ribonuclease A. As hepatocytes are known to possess a Na-H exchanger, the most straightforward interpretation of our data is that a mammalian Na-H exchanger has been expressed in the muscle fibre of an invertebrate.     

Orientation of spin labels attached to cross-bridges in contracting muscle fibres

Electron micrographs showing different cross-bridge orientations in different states of muscle fibres, and X-ray diffraction patterns indicating axial cross-bridge disorder in contracting muscle first suggested that force generation in the contracting muscle involved a change in orientation of the myosin heads that form cross-bridges between thick and thin filaments. This has been supported by subsequent work; the myosin molecule has the required flexibility for changes in orientation. The orientation of muscle tryptophans and of probes attached to the myosin heads of permeable muscle fibres depends on the state of the muscle. Recently, fluorescence polarization fluctuations and time-resolved X-ray diffraction patterns have suggested that cross-bridges of a contracting muscle can rotate. We have used electron paramagnetic resonance (EPR) spectroscopy to monitor the orientation of spin labels attached specifically to a reactive sulphydryl on the myosin heads in glycerinated rabbit psoas skeletal muscle. Previously, it has been shown that the paramagnetic probes are highly ordered in rigor muscle, with a nearly random angular distribution in relaxed muscle. We show here that during the generation of isometric tension, approximately 80% of the probes display a random angular distribution as in relaxed muscle while the remaining 20% are highly oriented at the same angle as found in rigor muscle. These findings indicate that a domain of the myosin head does not change orientation during the power stroke of the contractile interaction.     

Dephosphorylated SRp38 acts as a splicing repressor in response to heat shock

The cellular response to stresses such as heat shock involves changes in gene expression. It is well known that the splicing of messenger RNA precursors is generally repressed on heat shock, but the factors responsible have not been identified. SRp38 is an SR protein splicing factor that functions as a general repressor of splicing. It is activated by dephosphorylation and required for splicing repression in M-phase cells. Here we show that SRp38 is also dephosphorylated on heat shock and that this dephosphorylation correlates with splicing inhibition. Notably, depletion of SRp38 from heat-shocked cell extracts derepresses splicing, and adding back dephosphorylated SRp38 specifically restores inhibition. We further show that dephosphorylated SRp38 interacts with a U1 small nuclear ribonucleoprotein particle (snRNP) protein, and that this interaction interferes with 5'-splice-site recognition by the U1 snRNP. Finally, SRp38-deficient DT40 cells show an altered cell-cycle profile consistent with a mitotic defect; they are also temperature sensitive and defective in recovery after heat shock. SRp38 thus plays a crucial role in cell survival under stress conditions by inhibiting the splicing machinery.     

Intrinsic differences in the growth rate of early nerve fibres related to target distance

Target field innervation in the developing vertebrate nervous system coincides with the onset of important trophic interactions. Two factors that determine the timing of this event are the distance axons have to grow to reach their targets, which are known to vary, and the rate at which they grow. There have been few studies of axonal growth rate at this stage of development and no comparative study of the relationship between growth rate and target distance. Embryonic chick cranial sensory neurons are located in discrete ganglia and the distance axons have to grow to reach their targets is different for each ganglion, ranging from several hundred to several thousand microns. Here, I show that these neurons differ in their in vivo growth rates; neurons with more distant targets growing faster. In vitro, single isolated neurons from each of these populations grow at a similar rate to that observed in vivo, indicating that growth rate is an intrinsically determined property of neurons before they reach their targets.     

Hedgehog acts as a somatic stem cell factor in the Drosophila ovary

Secreted signalling molecules of the Hedgehog (Hh) family have many essential patterning roles during development of diverse organisms including Drosophila and humans. Although Hedgehog proteins most commonly affect cell fate, they can also stimulate cell proliferation. In humans several distinctive cancers, including basal-cell carcinoma, result from mutations that aberrantly activate Hh signal transduction. In Drosophila, Hh directly stimulates proliferation of ovarian somatic cells. Here we show that Hh acts specifically on stem cells in the Drosophila ovary. These cells cannot proliferate as stem cells in the absence of Hh signalling, whereas excessive Hh signalling produces supernumerary stem cells. We deduce that Hh is a stem-cell factor and suggest that human cancers due to excessive Hh signalling might result from aberrant expansion of stem cell pools.     

Hypervariable C-terminal domain of rab proteins acts as a targeting signal

Mammalian cells express many ras-like low molecular mass GTP-binding proteins (rab proteins) that are highly homologous to the Ypt1 and Sec4 proteins involved in controlling secretion in yeast. Owing to their structural similarity and to their variety, rab proteins have been postulated to act as specific regulators of membrane traffic in exocytosis and endocytosis, and rab5 has been shown to be involved in early endosome fusion in vitro. In agreement with their postulated functions, all rab proteins studied so far have been found in distinct subcompartments along the exocytic or endocytic pathways. To define the region mediating their specific localization, we transiently expressed rab2, rab5 and rab7 hybrid proteins in BHK cells, and determined their intracellular localization by immunofluorescence confocal microscopy and subcellular fractionation. Here we present evidence that the highly variable C-terminal domain contains structural elements necessary for the association of rab proteins with their specific target membranes in the endocytic pathway.     

Insulin stimulates sugar transport in giant muscle fibres of the barnacle

Insulin stimulates sugar transport in vertebrate skeletal muscle but the mechanism of insulin action is unknown. It has been reported that Na transport in giant muscle fibers of the barnacle (Balanus nubilis) is sensitive to insulin but no one has examined the sensitivity of sugar tansport to insulin in this preparation. We show here that insulin does, indeed, stimulate sugar transport in barnacle muscle. The great advantage of barnacle muscle over all other muscles used so far for investigating the mechanism of insulin action is its large size, which facilitates measurements on single cells and permits the experimenter to control the intracellular environment of the muscle fibre by the technique of internal dialysis. Using single muscle fibres it is possible to show that acceleration of sugar transport by insulin is associated with a fall in ionized Ca, a fall in cyclic AMP and a rise in cyclic GMP. Working with internally dialysed muscle fibres we find that insulin only increases sugar transport when the dialysis solution contains ATP. In the absence of insulin, sugar transport is dialysed muscle is increased by a rise in ionized Ca, a fall in cyclic AMP and, when the internal Ca is elevated, by a rise in cyclic GMP.     

'Square arrays' in the sarcolemma of human skeletal muscle fibres.

Fast twitch fibres of rat and rabbit show rectangular patterns of intramembrane particles in freeze-fracture preparations of the sarcolemma. These 'square arrays' are almost totally absent in the slow twitch coleus muscle of rat. I report here differences in the incidence of square arrays in human fetal and adult muscle and in different fibres within a human muscle. Square arrays probably classify fast and slow twitch fibres in freeze-fracture preparations of mixed muscles.