Philip W. Sheard

Changes in mitochondrial membrane potential during staurosporine-induced apoptosis in Jurkat cells

Cytochrome c release from mitochondria is central to apoptosis, but the events leading up to it are disputed. The mitochondrial membrane potential has been reported to decrease, increase or remain unchanged during cytochrome c release. We measured mitochondrial membrane potential in Jurkat cells undergoing apoptosis by the uptake of the radiolabelled lipophilic cation TPMP, enabling small changes in potential to be determined. The ATP/ADP ratio, mitochondrial and cell volumes, plasma membrane potential and the mitochondrial membrane potential in permeabilised cells were also measured. Before cytochrome c release the mitochondrial membrane potential increased, followed by a decrease in potential associated with mitochondrial swelling and the release of cytochrome c and DDP‐1, an intermembrane space house keeping protein. Mitochondrial swelling and cytochrome c release were both blocked by bongkrekic acid, an inhibitor of the permeability transition. We conclude that during apoptosis mitochondria undergo an initial priming phase associated with hyperpolarisation which leads to an effector phase, during which mitochondria swell and release cytochrome c.

Y-box factor YB1 controls p53 apoptotic function.

Nuclear localization and high levels of the Y-box-binding protein YB1 appear to be important indicators of drug resistance and tumor prognosis. YB1 also interacts with the p53 tumor suppressor protein. In this paper, we have continued to explore YB1/p53 interactions. We report that transcriptionally active p53 is required for nuclear localization of YB1. We go on to show that nuclear YB1 regulates p53 function. Our data demonstrate that YB1 inhibits the ability of p53 to cause cell death and to transactivate cell death genes, but does not interfere with the ability of p53 to transactivate the CDKN1A gene, encoding the kinase p21WAF1/CIP1 required for cell cycle arrest, nor the MDM2 gene. We also show that nuclear YB1 is associated with a failure to increase the level of the Bax protein in normal mammary epithelial cells after stress activation of p53. Together these data suggest that (nuclear) YB1 selectively alters p53 activity, which may in part provide an explanation for the correlation of nuclear YB1 with drug resistance and poor tumor prognosis.

Examination of intrafascicular muscle fiber terminations: Implications for tension delivery in series‐fibered muscles

Mammalian skeletal muscles with long fascicle lengths are predominantly composed of short muscle fibers that terminate midbelly with no direct connection to the muscle origin or insertion. The manner in which these short fibers terminate and transmit tension through the muscle to their tendons is poorly understood. We made an extensive morphological study of a series‐fibered muscle, the guinea pig sternomastoid, in order to define the full range of structural specializations for tension transmission from short fibers within this muscle. Terminations were examined in single fibers, teased small bundles of fibers, and in sections at both the light and electron microscopic level. In many cases, sites of fiber termination were defined by reactivity for the enzyme acetylcholinesterase, which also marks myotendinous junctions. Additionally, transport of the lipophilic fluorescent dye, DiI, or injection of Lucifer Yellow were used to visualize undisturbed fiber terminations in whole muscles using confocal and fluorescence microscopy. At the light microscopic level, we find that intrafascicularly terminating fibers end about equally often in either a long progressive taper, or in a series of small or larger blunt steps. Combinations of these two morphologies are also seen. However, when analyzed at higher resolution with confocal or electron microscopy, the apparently smooth progressive tapers appear also to be predominantly composed of a series of fine stepped terminations. Stepwise terminations in most cases join face‐to‐face with complementary endings of neighboring muscle fibers, some via an extended collagenous bridge and others at close interdigitating myomyonal junctions. These muscle‐to‐muscle junctions show many of the features of myotendinous junctions, including dense subsarcolemmal plaques in regions of myofibrillar termination and we suggest that they serve to pass tension from fiber to fiber along the longitudinal axis of the muscle. In addition, we observe regions of apparent side‐to‐side adhesion between neighboring fibers at sites where there is no apparent fiber tapering or structural specialization typical of myofibril termination. These sites show acetylcholinesterase reactivity, and large numbers of collagen fibers passing laterally from fiber to fiber. These latter connections seem most likely to be involved in lateral transmission of tension, either from fiber to fiber, or from fiber to endomysium. Overall, our results suggest that tension from intrafascicularly terminating fibers is likely to be passed along the muscle to the tendon using both in‐series and in‐parallel arrangements. The results are discussed in light of current theories of tension delivery within the series‐fibered muscles typical of large, nonprimate mammals. J. Morphol. 245:130–145, 2000

Nuclear localization of Y-box factor YB1 requires wild-type p53.

Nuclear localization and high levels of the Y-box binding protein YB1 appear to be important indicators of drug resistance and tumor prognosis. YB1 also interacts with the p53 tumor suppressor protein. In this paper, we explore a role for p53 in the nuclear localization of YB1. We report that various genotoxic stresses induce nuclear localization of YB1 in a small proportion of treated cells, but only in cells with wild-type p53. We go on to show directly that functional p53 is required for YB1 to translocate to the nucleus. Tumor-associated p53 mutants however are attenuated for YB1 nuclear localization as are mutants mutated in the proline-rich domain of p53. These data link the DNA-damage response of p53 to YB1 nuclear translocation. In addition, we find that YB1 inhibits p53-induced cell death and its ability to trans-activate promoters of genes involved in cell death signaling. Together these data suggest that some forms of p53 cause YB1 to accumulate in the nucleus, which in turn inhibits p53 activity. These results provide a possible explanation for the correlation of nuclear YB1 with drug resistance and poor prognosis in some tumor types, and for the first time implicate p53 in the process of nuclear translocation.

Developmental loss of NT-3 in vivo results in reduced levels of myelin-specific proteins, a reduced extent of myelination and increased apoptosis of Schwann cells

This work investigates the role of NT‐3 in peripheral myelination. Recent articles, based in vitro, propose that NT‐3 acting through its high‐affinity receptor TrkC may act to inhibit myelin formation by enhancing Schwann cell motility and/or migration. Here, we investigate this hypothesis in vivo by examining myelination formation in NT‐3 mutant mice. On the day of birth, soon after the onset of myelination, axons showed normal ensheathment by Schwann cells, no change in the proportion of axons which had begun to myelinate, and no change in either myelin thickness or number of myelin lamellae. However in postnatal day 21 mice, when myelination is substantially complete, we observed an unexpected reduction in mRNA and protein levels for MAG and P0, and in myelin thickness. This is the opposite result to that predicted from previous in vitro studies, where removal of an inhibitory NT‐3 signal would have been expected to enhance myelination. These results suggest that, in vivo, the importance of NT‐3 as a major support factor for Schwann cells (Meier et al., ( 1999 ) J Neurosci 19:3847‐3859) over‐rides its potential role as an myelin inhibitor, with the net effect that loss of NT‐3 results in degradation of Schwann cell functions, including myelination. In support of this idea, Schwann cells of NT‐3 null mutants showed increased expression of activated caspase‐3. Finally, we observed significant reduction in width of the Schwann cell periaxonal collar in NT‐3 mutant animals suggesting that loss of NT‐3 and resulting reduction in MAG levels may alter signaling at the axon‐glial interface.

Nerve Growth Factor mRNA Expression in the Regenerating Antler Tip of Red Deer (Cervus elaphus)

Deer antlers are the only mammalian organs that can fully regenerate each year. During their growth phase, antlers of red deer extend at a rate of approximately 10 mm/day, a growth rate matched by the antler nerves. It was demonstrated in a previous study that extracts from deer velvet antler can promote neurite outgrowth from neural explants, suggesting a possible role for Nerve Growth Factor (NGF) in antler innervation. Here we showed using the techniques of Northern blot analysis, denervation, immunohistochemistry and in situ hybridization that NGF mRNA was expressed in the regenerating antler, principally in the smooth muscle of the arteries and arterioles of the growing antler tip. Regenerating axons followed the route of the major blood vessels, located at the interface between the dermis and the reserve mesenchyme of the antler. Denervation experiments suggested a causal relationship exists between NGF mRNA expression in arterial smooth muscle and sensory axons in the antler tip. We hypothesize that NGF expressed in the smooth muscle of the arteries and arterioles promotes and maintains antler angiogenesis and this role positions NGF ahead of axons during antler growth. As a result, NGF can serve a second role, attracting sensory axons into the antler, and thus it can provide a guidance cue to define the nerve track. This would explain the phenomenon whereby re-innervation of the regenerating antler follows vascular ingrowth. The annual growth of deer antler presents a unique opportunity to better understand the factors involved in rapid nerve regeneration.

Neurotrophin‐3 null mutant mice display a postnatal motor neuropathy

This paper examines early postnatal development of the neuromuscular system in mice with a null mutation in the gene for neurotrophin‐3. We report that alpha‐motoneurons at first develop substantially normally, despite a known 15% deficit in their somal size [ Woolley et al. (1999)Neurosci. Lett., 272, 107–110.] and the absence of proprioceptive input [ Ernfors et al. (1994)Cell, 77, 503–512]. At birth, motor axons have extended into the muscle, forming normal‐looking neuromuscular junctions with focal accumulations of acetylcholine receptors. Detailed ultrastructural analysis does however, reveal subtle abnormalities at this time, particularly a decrease in the extent of occupancy of the postsynaptic site by nerve terminals, and a small but significant deficit in myofibre number. After the relative normality of this early neuromuscular development, there then occurs a catastrophic postnatal loss of motor nerve terminals, resulting in complete denervation of hindlimb muscles by P7. In systematic semi‐serial samples through the entire muscle endplate zones, no neuromuscular junctions can be found. Intramuscular axons are fragmented, as shown by both electron microscopic observations and neurofilament immunohistochemistry, and alpha‐bungarotoxin detection of acetylcholine receptors indicates dispersal of the junctional accumulation. At earlier times (postnatal days three and four) the terminal Schwann cells show ultrastructural abnormalities, and preliminary observations suggest marked disturbance of myelination. Based on comparison with other literature, the peripheral nerve degeneration seems unlikely to have arisen as a secondary effect of de‐afferentation. We discuss whether the neural degeneration is secondary to the disturbance of Schwann cell function, or due directly to a loss of neurotrophin‐3 based support of the motoneuron.

Intramuscular Force Transmission

The architectural form of skeletal muscle, the pattern of activity/usage between neighbouring fibres, and the pathways for lateral and lengthwise tension delivery are all of interest in understanding muscle function and dysfunction. We have attempted to contribute to understanding of intramuscular force transmission by investigating the functional relationships between coactive motor units, and by examining the detailed molecular and morphological features at sites of tension transfer. We found that tension delivery is modulated by interaction between active and inactive fibres, that many muscle fibre terminations feature structural coupling between fibres, and that sites of tension delivery feature a variety of proteins including acetylcholinesterase, NCAM, dystrophin and two splice variants of the alpha7 integrins. We conclude that structural and molecular pathways exist to deliver force within, along, and between muscle fibres, and that the quality/quantity of tension delivered from any single fibre is at least partly a consequence of whether its neighbouring fibres are synchronously coactive.

Alpha motoneurons are present in normal numbers but with reduced soma size in neurotrophin-3 knockout mice.

Neurotrophin-3 (NT-3) is essential for survival of proprioceptive and mechanoreceptor neurons, but its role in motoneuron development in vivo has seemed slight. Recent evidence however has suggested that NT-3 may be involved in motoneuron maturation. Here, we quantitatively assess the number and state of development of motoneurons within the lumbar lateral motor column (LLMC) of newborn NT-3 (-/-), (+/-) and (+/+) mutant mice. We find that the number of alpha motoneurons in the LLMC is the same in all genotypes, but soma size is significantly reduced in (-/-) mutants. This suggests that NT-3 is not required for normal production and survival of alpha motoneurons, but is essential for their full maturation and/or maintenance. We also confirm a previous report that gamma motoneurons are absent in NT-3 (-/-) mice.

Lifelong exercise and locally produced insulin-like growth factor-1 (IGF-1) have a modest influence on reducing age-related muscle wasting in mice

The age‐related loss of skeletal muscle mass and function is termed sarcopenia and has been attributed to a decline in concentrations of insulin‐like growth factor‐1 (IGF‐1). We hypothesized that constitutively expressed IGF‐1 within skeletal muscles with or without exercise would prevent sarcopenia. Male transgenic mice that overexpress IGF‐1 Ea in skeletal muscles were compared with wild‐type littermates. Four‐month‐old mice were assigned to be sedentary, or had access to free‐running wheels, until 18 or 28 months of age. In wild‐type mice, the mass of the quadriceps muscles was reduced at 28 months and exercise prevented such loss, without affecting the diameter of myofibers. Conversely, increased IGF‐1 alone was ineffective, whereas the combination of exercise and IGF‐1 was additive in maintaining the diameter of myofibers in the quadriceps muscles. For other muscles, the combination of IGF‐1 and exercise was variable and either increased or decreased the mass at 18 months of age, but was ineffective thereafter. Despite an increase in the diameter of myofibers, grip strength was not improved. In conclusion, our data show that exercise and IGF‐1 have a modest effect on reducing aged‐related wasting of skeletal muscle, but that there is no improvement in muscle function when assessed by grip strength.