Exploiting Bioenergetic Differences to Stop GVHD

Image: Flickr - Junichiro Aoyama
Bz-423, a mitochondrial F1F0-ATP synthase inhibitor, that has previously shown promise for the treatment of autoimmune disorders such as lupus, arthritis and psoriasis has now been shown to halt the progression of established graft-versus-host disease (GVHD) in mouse models of allogeneic bone marrow transplantation. GVHD, in which functional immune cells in the transplanted marrow mount an immunological attack on the recipient, is a common complication of allogeneic bone marrow transplantation that can cause severe organ damage and even be life-threatening. As well as demonstrating that Bz-423 can reduce GVHD clinical scores and improve survival in mice, researchers at the University of Michigan and the University of Florida have shed new light on the metabolism of alloreactive T cells.

Bz-423 structure
Resting lymphocytes meet their minimal demand for ATP using low rates of oxidative phosphorylation. Upon activation, normal lymphocytes meet the increased demand for ATP by dramatically increasing their rate of aerobic glycolysis which also helps to maintain high levels of antioxidants in activated cells. Data from the present study suggest that alloreactive T cells, on the other hand, rely heavily on increased oxidative phosphorylation to generate more ATP. This difference in energy generation is also seen in pathogenic T cells involved in autoimmune diseases and provides a mechanistic basis for the specific elimination of pathogenic cells by Bz-423, whilst preserving normal immune function.

The study is published in Science Translational Medicine.

Lycera Corporation has a program to develop orally bioavailable F1F0-ATP synthase inhibitors that exploit bioenergetic abnormalities in pathologically activated lymphocytes and result in the selective silencing of these cells.

The Right Tool for the Job?

Image: Flickr - Janne Moren
Tool compounds are used to explore the role of a specific protein in a biological context and – it goes without saying – that to obtain meaningful results in a complex situation, the tool compound should have appropriate potency and selectivity. The family of phospholipase C (PLC) enzymes play important regulatory roles and a small molecule inhibitor, U73122, has been extensively used to provide evidence for the involvement of PLCs in many cellular pathways. Recent reports, however, have questioned the selectivity of U73122 and scientists at the University of North Carolina and GlaxoSmithKline have now discovered that, even in its interaction with PLCs, U73122 may not be quite what it seems. When the team explored the effects of U73122 on human PLCs in cell-free micellar systems, they found that the compound actually increased the enzymatic activity of a number of isoforms in a concentration- and time-dependent manner. At micromolar concentrations, U713122 increased the activity of PLCβ3 by up to eight-fold, that of PLCγ1 by more than ten-fold, and that of PLCβ2 by around two-fold; PLCδ1 was neither activated nor inhibited.

U73122 structure
Activation of PLCβ3 was attenuated by competing nucleophiles, suggesting that activation involves covalent modification of the protein by the reactive maleimide group of U73122; the analogous succinimide, U73343, was not effective as an activator. Involvement of specific cysteine residues in the protein was demonstrated by LC/MS/MS experiments. Although N-ethyl maleimide (NEM) itself did not activate PLCβ3, excess NEM attenuated the U73122-mediated activation in a concentration-dependent manner. The authors propose an activation model in which U73122 irreversibly binds to multiple cysteine residues on PLCβ3 and acts as either a lipid anchor or interfacial recognition site for the enzyme, facilitating adsorption to the substrate interface (i.e. the micelle surface). The protein-linked U73122 increases the rate of lipase activity by keeping the enzyme in close proximity to substrate which is held in the membrane.

The study, which is published in the Journal of Biological Chemistry, provides strong evidence that U73122 activates PLC enzymes in cell-free systems, in contrast to its ‘established’ role as a specific inhibitor of this family. The authors suggest that U73122 may have opposing effects on cytosolic and membrane-bound enzymes and/or may modify other cellular nucleophiles and advise great caution when forming hypotheses based on the observed effects of U73122 in cellular systems.

Barring Leukocytes May Reduce Multiple Sclerosis Symptoms

Image: Flickr - Darren D
Multiple sclerosis (MS), believed to be an immune-mediated disorder, is the most common disabling condition of the central nervous system (CNS) affecting young adults. Infiltration of leukocytes into the brain, helped by upregulation of matrix metalloproteinases (MMPs) which cleave components of the extracellular matrix, plays a significant role in causing the demyelination and axonal degeneration associated with MS.

Synthesis of MMPs is regulated by extracellular matrix metalloproteinase inducer (EMMPRIN, CD147), a multifunctional member of the immunoglobulin superfamily, and researchers at the Hotchkiss Brain Institute, University of Calgary have shown that EMMPRIN levels are significantly increased in the brain of MS patients, particularly in plaque-containing regions. The team also showed that EMMPRIN levels are increased in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EMMPRIN was upregulated on peripheral leukocytes before the appearance of MS-like symptoms and on infiltrating leukocytes and resident cells within the CNS once clinical symptoms had developed. Treating the mice with an anti-EMMPRIN antibody reduced MMP activity, infiltration of leukocytes into the CNS, and the severity of MS-like symptoms. To be effective, the antibody had to be administered at the onset of clinical signs, a time that is typically associated with significant infiltration of leukocytes into the CNS.

The study, which is published in the Journal of Neuroscience, suggests that blocking the activity of EMMPRIN may be a novel way to treat MS.

Single Catastrophic Event Can Lead to Cancer

Image: Flickr - Mohamed Hussain
Development of cancer is conventionally viewed as a gradual process, taking years to accumulate multiple point mutations and chromosomal rearrangements, and progressing through increasingly malignant phenotypes. New research by a team at the Wellcome Trust Sanger Institute has shown that, in some cases, cancer can result from a single catastrophic event involving tens to hundreds of genomic rearrangements. Using advanced DNA sequencing techniques, the team found that 2-3% of cancer samples, across many common subtypes, had dramatic structural changes affecting highly localised regions of one or more chromosomes that could not be explained using standard models of DNA damage. This type of damage was especially common in bone cancers, where around 25% of samples showed signs of chromosomal crisis.

The team have proposed that such extensive damage is likely to occur when the chromosomes are condensed for mitosis and could be caused by ionising radiation or linked to telomere attrition. If the cell attempts to repair such extensive damage, the evidence suggests that the repair goes badly wrong, resulting in a genome that is riddled with mutations. In most cases, such haphazard repairs would be detrimental to the cell’s ability to survive and divide but in some cases can amplify cancer genes or inactivate tumour suppressor genes. It is remarkable that the cell can not only survive such a cataclysmic event but can emerge with a selective advantage.

The study is published in the journal, Cell.

A Closer Look at the Immune Response to Cancer Cells

Leukocytes: Friend or Foe to Transformed Cells? Image of a neutrophil (red) gliding over a doublet of transformed mucus-producing cells
Last week it was reported that a daily (low) dose of aspirin can significantly reduce the risk of dying from a variety of cancers, and a study published in PloS Biology now opens a new window on the role of inflammation in cancer.

Although the host immune system is known to have conflicting roles in cancer initiation and progression, both acting as a surveillance and elimination system but also assisting expansion and metastatic spread of tumours, very little is known about the very early role of the immune system in cancer. Using zebrafish larvae, researchers at the University of Bristol, the University of Manchester and the FIRC Institute of Molecular Oncology in Milan have now been able to observe, for the first time, how oncogene-transformed cells in the skin co-opt the innate immune system to promote their growth from the very earliest stages of development. The team exploited the translucency of the larvae to obtain live images of the earliest interactions between the cancer cells and the immune environment. Using larvae with fluorescently tagged leukocytes, the team were able to observe recruitment of neutrophils and macrophages to oncogene-transformed melanocytes or mucus-secreting cells. As well as engulfment of the transformed cells, the team saw many examples of cytoplasmic tethers linking the two cell types.

They discovered that a key attractant for the leukocytes was hydrogen peroxide. Both the transformed cells themselves and otherwise healthy neighbouring cells were found to produce hydrogen peroxide, which is also a key molecule that recruits neutrophils to a wound. Blocking the synthesis of hydrogen peroxide prevented recruitment of immune cells and reduced the number of transformed cells, suggesting that immune cells may provide trophic support to the transformed cells just as they promote repair at a site of tissue injury. Unlike the case of wound healing, however, where the inflammatory response resolves, the inflammatory response to transformed cells seems to amplify and progress towards a chronic inflammatory state similar to that seen in chronic non-healing wounds.

Cold Receptors Bring Tears to the Eyes

Image: Wikimedia Commons
Transient receptor potential (TRP) ion channels mediate a variety of sensations including thermal stimuli. The channels also react to multiple ligands: for example, TRPV1 channels respond to heat as well as to ligands which elicit a hot sensation such as capsaicin, whereas TRPM8 channels respond to cold and also to ligands which elicit a cool sensation such as menthol. Since TRP channels are expressed on sensory neurons, a number of groups are developing selective channel modulators for the treatment of pain.

Researchers led by a team at the Instituto de Neurociencias de Alicante have now shown that TRPM8 channels in the eye also regulate basal tear secretion. In studies in mice, the researchers found that cooling of the eye surface by 1-2°C by evaporation of the tear film can induce production of more tears by stimulation of TRPM8 in nerve endings in the cornea. In mice lacking TRPM8, the cornea was insensitive to cooling and the basal rate of tear production was greatly reduced. Tear production caused by exposure to irritants which is mediated by other channels such as TRPV1 was, however, unaffected in the TRPM8 deficient mice. In normal mice, tear secretion could also be decreased by raising the temperature of the cornea to 33-36°C. Tear production in humans is also regulated by cold – the basal rate of tear production is significantly lower at 43°C than at 18-20°C.

The study, which is published in the journal Nature Medicine, indicates that TRPM8 contributes to the regulation of basal tear flow and opens new possibilities for the treatment of dry eye syndrome by increasing tear secretion. Dry mucosal surfaces, including dry eye syndrome, are a common problem, particularly for the elderly with up to one third of people over the age of sixty five estimated to have dry eyes.

Blocking Faulty Replication Could Boost Response to Chemotherapy

Image: Flickr - Mark Cummins
Many chemotherapy drugs, including cisplatin, cause damage to DNA and kill cancer cells by interfering with DNA replication and cell division. The damage activates cellular DNA repair mechanisms but, if the damage is too extensive, the cell undergoes apoptosis. Unfortunately, although the initial response to cisplatin is generally good, the majority of tumours will eventually develop resistance to the drug. Resistance can develop when the cell is able to replicate DNA through damaged regions using a translesion synthesis (TSL) DNA polymerase. This type of DNA replication is highly error-prone, introducing mutations into the DNA which can drive drug resistance. Suppressing the ability of tumour cells to replicate damaged DNA using the translesion synthesis DNA polymerase, Polζ has been shown to block resistance to cisplatin in human cancer cells grown in culture and now, in two papers published in PNAS, researchers at the Massachusetts Institute of Technology have shown that the approach also works in mice.

The first paper describes a tumour transplantation approach to examine the effect of impaired translesion DNA synthesis on cisplatin response in aggressive late-stage lung cancers. The researchers used RNA interference to reduce levels of Rev3, an essential component of Polζ, and showed that a 60-70% reduction doubled survival time in cisplatin-treated animals. The team also showed that Rev3-deficient cells showed reduced cisplatin-induced mutations which have been suggested to contribute to secondary malignancies following chemotherapy.

In the second study, the researchers used a mouse model of B-cell lymphoma to show that suppressing Rev1, an essential TSL scaffold protein and dCMP transferase, inhibits both cisplatin- and cyclophosphamide-induced mutagenesis. By performing repeated cycles of tumor engraftment and treatment, the team were also able to show that Rev1 plays a critical role in the development of acquired cyclophosphamide resistance.

The studies show that chemotherapy can not only select for drug-resistant populations of tumour cells but can also directly promote the acquisition of resistance-causing mutations, suggesting that blocking translesion DNA polymerases may have dual anticancer effects by both increasing the sensitivity of tumours to chemotherapy as well as reducing the potential for emergence of drug resistance during treatment. The next challenge will be to identify inhibitors of the translesion DNA polymerases.

Stressful ‘Lifestyle’ is Bad for Neurons

Image: Flickr - greenmelinda
Parkinson’s disease is characterised by loss of dopaminergic neurons in the area of the midbrain known as the substantia nigra. Although mitochondrial stress – an accumulation of damaging superoxide and free radicals – is believed to be the cause of cell death, it is not understood why this subset of neurons is especially vulnerable.

Researchers at Northwestern University have now suggested a possible answer: these neurons have an inherently stressful ‘lifestyle’. The cells in the substantia nigra act as pacemakers, releasing rhythmic bursts of dopamine. This activity is accompanied by an influx of calcium ions which must then be pumped back out of the cell in an energy-demanding process. The inflow of calcium ions is not essential for pacemaking activity so, if the energy needed to pump calcium ions out of the cell is adding extra stress, blocking the influx of calcium should help to alleviate this. Using mice engineered to express a redox-sensitive fluorescent protein in their mitochondria, the team showed that the opening of L-type calcium channels during normal pacemaking activity created an oxidant stress that was specific to dopaminergic cells of the substantia nigra. The oxidative stress, in turn, caused a defensive mild mitochondrial depolarization or uncoupling.

Although most cases of Parkinson’s disease have no known genetic cause, loss-of-function DJ-1 (PARK7) mutations can cause early-onset Parkinson’s disease in humans and transgenic mice lacking DJ-1 also show damage to dopaminergic cells in the substantia nigra. Knocking out DJ-1 down-regulates expression of two uncoupling proteins and increases oxidation of mitochondrial matrix proteins in dopaminergic neurons of the substantia nigra. Treatment of the transgenic animals with the L-type calcium channel blocker, isradipine, was found to protect the dopaminergic cells of the substantia nigra from oxidative damage.

The study, which is published in the journal Nature, builds on previous studies linking calcium channel blockade with protective effects in Parkinson’s disease.

A clinical trial is currently underway to examine the safety, tolerability and efficacy of isradipine – which is already approved for the treatment of high blood pressure – in patients with Parkinson’s disease. The hope is that the drug will slow disease progression and allow a broader window for existing symptomatic treatments.

New Insight into Reducing Stroke Damage

Image: Flickr - jpre86
Stroke is the third most common cause of death in the developed world and is also the leading cause of serious long-term adult disability; many survivors never recover sufficient function to live independently. Although rapid intervention to restore blood flow to the affected area can improve outcomes, the brain has limited capacity for repair and there is currently no treatment that helps recovery. The zone immediately surrounding the damaged area is critically important for recovery since motor and sensory neurons in this region can make new connections and compensate for those killed by the stroke.

L-655,708 structure
Immediately after a stroke, tonic inhibition in the affected area increases to reduce excitability and limit the extent of the damage, but this increased tonic inhibition also has the effect of reducing plasticity in surrounding areas. Researchers at UCLA and the University of Otago have now shown that the increased tonic inhibition can persist for weeks and eventually hinder recovery. In experimentally induced stroke in mice, tonic neural inhibition was found to be increased in the area surrounding the stroke damage and shown to be mediated by extrasynaptic GABAA receptors. After a stroke in the motor cortex, six weeks treatment with L-655,708, a subtype-selective inverse agonist of the α5-subunit-containing extrasynaptic GABAA receptor, restored tonic inhibition to pre-stroke levels and led to a sustained improvement of motor function. In keeping with a protective role of tonic inhibition immediately after a stroke, the treatment was only effective if delayed until three days after the stroke; initiating treatment too early increased the damage caused by the stroke.

The results suggest that reduction of tonic inhibition by reducing extrasynaptic GABAA receptor function could be beneficial in promoting recovery after stroke and possibly other brain traumas. A treatment that is effective following delayed administration would offer a significant advantage over existing interventions which must be carried out within a few hours of the stroke occurring.

The study is published in the journal Nature.

No More (Involuntary) Laughing and Crying

venetian mask
Image: Flickr - gnuckx
Pseudobulbar affect (PBA) occurs in people with brain injury or underlying neurological conditions such as multiple sclerosis, amyotrophic lateral sclerosis and Parkinson’s disease and is characterised by unpredictable and uncontrollable fits of laughing or crying that may not reflect the individual’s underlying mood or may be inappropriate to the situation. Because many people feel embarrassed by their symptoms, PBA can have a significant effect on quality of life since sufferers tend to withdraw from social and professional activities. So far, there has been no specific treatment but, last week, the FDA approved Nuedexta™ as the first treatment of PBA.

Nuedexta™ is a combination of dextromethorphan, a component of some over-the-counter cough mixtures and the antiarrhythmic agent, quinidine, which acts as a metabolic inhibitor, allowing effective concentrations of dextromethorphan to be achieved.

Dextromethorphan acts on sigma-1 and NMDA receptors in the brain, although exactly how it exerts its therapeutic effects in patients with PBA is not known. Nuedexta™ has been shown to be safe and effective in patients with multiple sclerosis and amyotrophic lateral sclerosis but not in other groups of patients who may experience PBA, such as those with Alzheimer’s disease.

Nuedexta™ was developed by Avanir Pharmaceuticals, Inc. and is expected to be available by prescription in the US during the first quarter of 2011.