Rory Curtis

Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation but Does Not Alter Cell Survival following DNA Damage

ABSTRACT Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells.

Retrograde axonal transport of LIF is increased by peripheral nerve injury: Correlation with increased LIF expression in distal nerve

Leukemia inhibitory factor (LIF) is a cytokine that affects the survival and differentiation of certain neuronal populations in vitro. To identify LIF-responsive neurons in the adult rat, we have demonstrated retrograde axonal transport of 125I-LIF to sensory and motor neurons. The accumulation of 125I-LIF by both cell types was significantly increased by prior sciatic nerve crush. Retrograde transport of 125I-LIF was inhibited by excess unlabeled LIF but not by related cytokines, indicating a specific receptor-mediated mechanism. Northern blot analysis revealed LIF expression in peripheral nerve that was increased in distal segments after axotomy. The correlation between LIF expression and increased retrograde transport following injury suggests that LIF plays a role in peripheral nerve regeneration.

Distinct Subcellular Localization of Different Sodium Channel α and β Subunits in Single Ventricular Myocytes From Mouse Heart

Background—Voltage-gated sodium channels composed of pore-forming &agr; and auxiliary &bgr; subunits are responsible for the rising phase of the action potential in cardiac muscle, but their localizations have not yet been clearly defined. Methods and Results—Immunocytochemical studies show that the principal cardiac &agr; subunit isoform Nav1.5 and the &bgr;2 subunit are preferentially localized in intercalated disks, identified by immunostaining of connexin 43, the major protein of cardiac gap junctions. The brain &agr; subunit isoforms Nav1.1, Nav1.3, and Nav1.6 are preferentially localized with &bgr;1 and &bgr;3 subunits in the transverse tubules, identified by immunostaining of &agr;-actinin, a cardiac z-line protein. The &bgr;1 subunit is also present in a small fraction of intercalated disks. The recently cloned &bgr;4 subunit, which closely resembles &bgr;2 in amino acid sequence, is also expressed in ventricular myocytes and is localized in intercalated disks as are &bgr;2 and Nav1.5. Conclusions—Our results suggest that the primary sodium channels present in ventricular myocytes are composed of Nav1.5 plus &bgr;2 and/or &bgr;4 subunits in intercalated disks and Nav1.1, Nav1.3, and Nav1.6 plus &bgr;1 and/or &bgr;3 subunits in the transverse tubules.

Differential role of the low affinity neurotrophin receptor (p75) in retrograde axonal transport of the neurotrophins

The receptor mechanisms mediating the retrograde axonal transport of the neurotrophins have been investigated in adult rats. We show that transport of the TrkB ligands NT-4 and BDNF to peripheral neurons is dependent on the low affinity neurotrophin receptor (LNR). Pharmacological manipulation of LNR in vivo using either an anti-LNR antibody or a soluble recombinant LNR extracellular domain completely blocked retrograde transport of NT-4 and BDNF to sensory neurons, while having minimal effects on the transport of NGF in either sensory or sympathetic neurons. Furthermore, in mice with a null mutation of LNR, the transport of NT-4 and BDNF, but not NGF, was dramatically reduced. These observations demonstrate a selective role for LNR in retrograde transport of the various neurotrophins from distinct target regions in vivo.

Aging networks in Caenorhabditis elegans: AMP‐activated protein kinase (aak‐2) links multiple aging and metabolism pathways

Molecular genetics in lower organisms has allowed the elucidation of pathways that modulate the aging process. In certain instances, evolutionarily conserved genes and pathways have been shown to regulate lifespan in mammals as well. Many gene products known to affect lifespan are intimately involved in the control of energy metabolism, including the fuel sensor AMP‐activated protein kinase (AMPK). We have shown previously that over‐expression of an AMPK α subunit in Caenorhabditis elegans, designated aak‐2, increases lifespan. Here we show the interaction of aak‐2 with other pathways known to control aging in worms. Lifespan extension caused by daf‐2/insulin‐like signaling mutations was highly dependent on aak‐2, as was the lifespan extension caused by over‐expression of the deacetylase, sir‐2.1. Similarly, there was partial requirement for aak‐2 in lifespan extension by mitochondrial mutations (isp‐1 and clk‐1). Conversely, aak‐2 was not required for lifespan extension in mutants lacking germline stem cells (glp‐1) or mutants of the eating response (eat‐2). These results show that aging is controlled by overlapping but distinct pathways and that AMPK/aak‐2 represents a node in a network of evolutionarily conserved biochemical pathways that control aging.

Neuronal Injury Increases Retrograde Axonal Transport of the Neurotrophins to Spinal Sensory Neurons and Motor Neurons via Multiple Receptor Mechanisms

We investigated the retrograde axonal transport of 125I-labeled neurotrophins (NGF, BDNF, NT-3, and NT-4) from the sciatic nerve to dorsal root ganglion (DRG) sensory neurons and spinal motor neurons in normal rats or after neuronal injury. DRG neurons showed increased transport of all neurotrophins following crush injury to the sciatic nerve. This was maximal 1 day after sciatic nerve crush and returned to control levels after 7 days. 125I-BDNF transport from sciatic nerve was elevated with injection either proximal to the lesion or directly into the crush site and after transection of the dorsal roots. All neurotrophin transport was receptor-mediated and consistent with neurotrophin binding to the low-affinity neurotrophin receptor (LNR) or Trk receptors. However, transport of 125I-labeled wheat germ agglutinin also increased 1 day after sciatic nerve crush, showing that increased uptake and transport is a generalized response to injury in DRG sensory neurons. Spinal cord motor neurons also showed increased neurotrophin transport following sciatic nerve injury, although this was maximal after 3 days. The transport of 125I-NGF depended on the expression of LNR by injured motor neurons, as demonstrated by competition experiments with unlabeled neurotrophins. The absence of TrkA in normal motor neurons or after axotomy was confirmed by immunostaining and in situ hybridization. Thus, increased transport of neurotrophic factors after neuronal injury is due to multiple receptor-mediated mechanisms including general increases in axonal transport capacity.

Neurotrophin-3 Administration Attenuates Deficits of Pyridoxine-Induced Large-Fiber Sensory Neuropathy

Chronic treatment of adult rats for 2–3 weeks with high doses of pyridoxine (vitamin B6) produced a profound proprioceptive loss, similar to that found in humans overdosed with this vitamin or treated with the chemotherapeutic agent cisplatin. Pyridoxine toxicity was manifest as deficits in simple and precise locomotion and sensory nerve function and as degeneration of large-diameter/large-fiber spinal sensory neurons. As assessed quantitatively in a beam-walking task and by EMG recording of H waves evoked by peripheral nerve stimulation, coadministration of the neurotrophic factor neurotrophin-3 (NT-3; 5–20 mg · kg−1 · d−1, s.c.) during chronic pyridoxine treatment largely attenuated the behavioral and electrophysiological sequelae associated with pyridoxine toxicity. Furthermore, NT-3 administration prevented degeneration of sensory fibers in the dorsal column of the spinal cord. These data are consistent with the evidence that NT-3 is a target-derived neurotrophic factor for muscle sensory afferents and suggest that pharmacological doses of NT-3 may be beneficial in the treatment of large-fiber sensory neuropathies.

Truncated TrkB mediates the endocytosis and release of BDNF and neurotrophin-4/5 by rat astrocytes and Schwann cells in vitro

Binding and cross-linking studies with radiolabeled neurotrophins demonstrate that cultured rat hippocampal astrocytes lack full-length TrkB, but do express high levels of truncated TrkB (tTrkB). In astrocytes and Schwann cells, tTrkB appears to have the novel function of mediating the endocytosis of neurotrophins into an acid-stable, Triton X-100 resistant intracellular pool that is released back into the medium in a temperature-dependent manner. Chloroquine treatment, trichloroacetic acid solubility, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that when incubated with astrocytes or Schwann cells for at least 48 h neither the intracellular nor the released neurotrophins were significantly degraded. The endocytosis and release of neurotrophins may represent a novel mechanism whereby neuroglia can regulate the local concentration of these neurotrophic factors for extended periods of time.

Ageing and metabolism: drug discovery opportunities

There has recently been significant progress in our understanding of the mechanisms that regulate ageing, and it has been shown that changes in single genes can dramatically extend lifespan and increase resistance to many diseases. Furthermore, many of these genes belong to evolutionarily conserved pathways that also control energy metabolism. In this review, we describe the shared molecular machinery that regulates ageing and energy metabolism. Although drugs to slow ageing face severe regulatory hurdles, it is likely that an understanding of ageing pathways will help to identify novel drug targets to treat metabolic disorders and other age-related diseases.

Chapter 4 Receptor mediated retrograde axonal transport of neurotrophic factors is increased after peripheral nerve injury

Publisher Summary Receptor-mediated retrograde axonal transport to sensory neurons of the dorsal root ganglia (DRG) has been demonstrated for the neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4), as well as the neuroactive cytokines, ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF). The receptor-mediated nature of these transport events has been demonstrated by a pharmacological approach. Increased transport of CNTF after peripheral nerve lesion may be an intrinsically programmed cellular mechanism for increasing the accumulation of much needed neurotrophic factors after neuronal damage. Further, injured or otherwise compromised neurons may show a selective responsiveness to neurotrophic factors by virtue of their increased retrograde transport. This is of significance when considering therapeutic intervention with pharmacological doses of neurotrophic factors for peripheral neuropathies. It is necessary to determine how the increased retrograde flow of neurotrophic signals is processed and what molecular mechanisms are operable in the transduction of these retrograde signals.