Common Pathway for Neurodegenerative Diseases

Image: Flickr - A_of_DooM
Nitrosylation of proteins is emerging as a key post-translational modification important in both normal physiology and a wide spectrum of diseases, including neurodegenerative diseases. Physiological levels of nitric oxide (NO) can be neuroprotective, in part at least, by inhibiting caspase activity, but excess NO production leads to activation of cell death signalling cascades involved in many neurodegenerative disorders. Neuronal cell injury and death, which are prominent features of disorders such as Alzheimer’s, Huntington’s, and Parkinson’s diseases, are often mediated by the caspase family of cysteine proteases. Caspase activity is inhibited by S-nitrosylation and is also regulated by inhibitors of apoptosis such as X-linked inhibitor of apoptosis (XIAP) which associates with active caspases and represses their catalytic activity. XIAP also functions as an E3 ubiquitin ligase, targeting caspases for degradation by the proteasome.

A team of scientists led by Sanford-Burnham researchers have now discovered a new twist in caspase regulation. They showed that S-nitrosylation of XIAP (forming SNO-XIAP) inhibits the protein’s E3 ligase and antiapoptotic activity and also found that XIAP can be transnitrosylated by SNO-caspase but not vice versa. They found significant amounts of SNO-XIAP, but not SNO-caspase, in the brains of individuals with neurodegenerative diseases, suggesting that SNO-XIAP contributes to neuronal injury or death. The team hope that their study, which is published in the journal Molecular Cell, might lead to better biomarkers and earlier diagnosis for neurodegenerative diseases.

Potential New Drugs for Epilepsy

Image: Flickr - Stephan Sachs
Over 30% of the 50 million people who are affected by epilepsy do not have their seizures adequately controlled, even with the best available medicines. The 29 amino acid neuropeptide, galanin, is a potent endogenous anticonvulsant that activates galanin receptors type 1 (GalR1) and type 2 (GalR2) and a number of groups, including researchers at the Scripps Research Institute, have been trying to develop drugs that mimic the effects of galanin as novel anticonvulsants. Galnon is an example of a systemically active nonpeptide galanin receptor ligand with affinity for the three galanin receptors which has been shown to reduce seizures in animal models. The Scripps team have now identified a compound that acts appears to act as a selective positive allosteric modulator of the galanin receptor type 2 (GalR2) which they hope will have a reduced potential for side effects compared with galnon. The compound, CYM2503, potentiated the effects of galanin in cells stably expressing the GalR2 receptor, but had no detectable affinity for the galanin binding site. In rodent models of epilepsy, intraperitoneal administration of CYM2503 increased the time to seizure, reduced the duration of seizures, and increased survival rate at 24 h.

The study is published in the Proceedings of the National Academy of Sciences.

Role for MMPs in Huntington’s Disease

Microscope image of a neuron with inclusion (stained orange) caused by HD, image width 250µm. Source: Wikipedia
Huntington’s disease is an inherited neurodegenerative disorder associated with mutations in the huntingtin gene on human chromosome 4. Although the functions of normal huntingtin protein are not entirely clear, it is known that abnormal huntingtin (mutantHtt, or mHtt) – and especially small proteolytic fragments of the protein – are toxic to neurons, particularly those in the striatum and cortex. Previous studies into the cleavage of huntingtin have focussed on the role of the cysteine protease families of caspases and calpains, but scientists at the Buck Institute for Age Research have now discovered that a metalloprotease also cleaves huntingtin into highly toxic fragments.

The team used a set of small interfering RNA (siRNA) pools targeting the 514 known and predicted human protease genes to identify those proteases involved in the cleavage of huntingtin. Eleven proteases were found to alter huntingtin fragment accumulation, and knockdown of the nine which are expressed in striatal cells significantly reduced huntingtin-mediated striatal cell death in a cellular toxicity screen. Amongst the proteases associated with huntingtin cleavage were three metalloproteases, MMP-10, MMP-14 and MMP-23B. Subsequent experiments showed that only MMP-10 directly cleaves huntingtin, suggesting that knockdown of MMP-10 reduces toxicity by directly altering proteolysis of huntingtin whereas knockdown of MMP-14 or MMP-23B modulates toxicity indirectly through proteolysis of cytokines or components of the extracellular matrix. Whilst matrix metalloproteases are generally thought to be secreted as proenzymes which are processed to the active forms extracellularly, MMP-10 was found to be activated inside the cell and to co-localise with huntingtin, suggesting that cleavage may occur intracellularly.

The study, which is published in the journal Neuron, suggests a role for matrix metalloproteases in the progression of Huntington’s disease and that development of inhibitors of MMP-10 may be a useful therapeutic strategy.

Illuminating the Link between Bone and Metabolism

Bone Tree
Image: Flickr – Livin-Lively
Two back-to-back studies published in the July 23rd issue of Cell, one from Columbia University Medical Center and the other from Johns Hopkins researchers, further the hypothesis that metabolic control and bone remodelling are inextricably linked. Both studies point to osteocalcin, a hormone released by bone, as a key mediator of this link.

The Johns Hopkins study used a conditional knock-out in mice to specifically suppress the expression of the insulin receptor in osteoblasts, the bone-forming cells of the skeletal system. As the mutant mice aged they became fat, had elevated blood sugar, and were glucose intolerant and resistant to insulin, mirroring the picture of diabetes in humans. The researchers found that the mutant mice had fewer osteoblasts, reduced bone formation and lower levels of circulating undercarboxylated osteocalcin (the active form of the hormone). The study showed that signalling via the insulin receptor in osteoblasts suppressed Twist2, an inhibitor of osteoblast development, and enhanced expression of osteocalcin, a mediator of insulin sensitivity and secretion.

The Columbia study links the complete bone remodelling process to energy regulation. Osteocalcin is released from osteoblasts predominantly in an inactive, carboxylated form. The researchers demonstrated that insulin signalling in osteoblasts stimulates release of inactive osteocalcin and activates osteoclasts, which activate the osteocalcin via decarboxylation in a bone-resorption-dependent manner.

The studies clearly have potential impact on human therapy, although significant questions remain. As yet the receptor for undercarboxylated osteocalcin is unknown, so the mechanism by with the hormone stimulates insulin release is unclear. Further work will be necessary to understand the interplay between skeletal- and metabolic-homeostasis in humans.

The two papers are previewed in Cell.

PPARγ– A New Twist in the Tale

Image: Flickr - alexdecarvalho
Obesity and related disorders such as diabetes have reached epidemic proportions. Although the anti-diabetic thiazolidinediones (glitazones) are effective insulin sensitizers, some members of the class have been withdrawn or had their use restricted because of safety concerns. Increased responsiveness to insulin is believed to be mediated by activation of the nuclear receptor, PPARγ but differences in clinically important side effects suggest subtle differences in pharmacology, even amongst full agonists.

Researchers at the Scripps Research Institute and the Dana-Farber Cancer Institute at Harvard University have now shown that cyclin-dependent kinase 5 (Cdk5) in adipose tissue is activated in obese mice fed a high-fat diet, resulting in phosphorylation of PPARγ. This has no effect on the adipogenic capacity of PPARγ but does alter the expression of a large number of obesity-related genes, including a reduction in expression of the insulin-sensitizing adipokine, adiponectin. Phosphorylation of PPARγ by Cdk5 was blocked both in vitro and in vivo by the full agonist, rosiglitazone, and by the partial agonist, MRL-24, leading to increased adiponectin production. The anti-diabetic effect of rosiglitazone in obese patients was also found to be closely associated with inhibition of PPARγ phosphorylation, suggesting that this may be a mechanism of insulin resistance. The authors of the study, which is published in the journal Nature, suggest that drugs that inhibit PPARγ phosphorylation by Cdk5, without necessarily activating the receptor, may provide an improved generation of anti-diabetic drugs.

Arsenic Gets Nanotechnology Treatment

arsenic trioxide model
Molecular model of arsenic trioxide as found in the mineral arsenolite. Source: Wikipedia – Ben Mills
Despite its reputation as a poison, arsenic has long been used in Chinese medicine and, more recently, arsenic trioxide has been successfully used to treat acute promyelocytic leukaemia (APL). The drug has poor activity against solid tumours, however, probably because it is rapidly excreted. Attempting to address this, researchers at Northwestern University have packaged arsenic trioxide in nanoparticles.

These nanoparticles, termed nanobins, are composed of nanoparticulate arsenic trioxide encapsulated in liposomes. A second chemical layer provides protection for both the cargo and normal cells until the particle reaches its target. The nanoparticles concentrate at their target, as a consequence of the leaky blood vessels that characterise solid tumours, and release their toxic payload.

In the current study, published in Clinical Cancer Research, the researchers investigated the activity of arsenic nanobins against a panel of human breast cancer cell lines. Although less cytotoxic than free arsenic trioxide in vitro, the nanobins had dramatically enhanced efficacy in an in vivo model of triple-negative breast cancer. The scientists observed reduced plasma clearance, increased tumour uptake and induction of tumour cell apoptosis for the nanobins.

Triple negative breast cancer, in which the receptors for oestrogen, progesterone and Her2 are absent, is an aggressive cancer that often responds poorly to conventional chemotherapy. There is a high risk of metastatis and survival rates are low. Although at an early stage, the researchers anticipate that the nanobin technology could provide the means to increase the efficacy of a number of cytotoxic drugs against a range of tumours, whilst reducing general toxicity.

Arctic Genes Provide Live Vaccines

Image: Flickr - carulmare
For decades, temperature-sensitive viruses have been generated by repeated passage at low temperatures and used as live vaccines against diseases such as polio and influenza. Typically, live vaccines against bacterial diseases have used strains with attenuated virulence generated by in vitro passage or deliberate mutagenesis.

Canadian and US scientists have now generated temperature-sensitive bacteria by substituting essential genes in pathogenic bacteria with counterparts from cold-loving C. psychrerythraea bacteria usually found in the subzero temperatures of the Arctic. These cold-adapted bacteria cannot survive at normal body temperatures and the temperature-sensitivity is transferred to the recipient bacteria, in this case F. novicida, which is normally highly virulent in mice. Out of nine temperature-sensitive strains of F. novicida, five were unable to revert to temperature-resistant forms at detectable levels. A temperature-sensitive vaccine is able to take advantage of the difference in temperature between the cooler tissues near the body surface and the warmer body core: the bacteria can grow at sites close to the skin but not at sites deep within the body. When mice were vaccinated s.c. near the tail with one of the temperature-sensitive strains of F. novicida, the bacteria grew close to the site of infection but did not spread to the spleen and the mice were afforded protection against a later lethal intranasal challenge with wild-type bacteria. As well as offering the possibility of live vaccines against important human pathogens such as S. enteric and M. tuberculosis, temperature-sensitive bacterial variants may also allow the biochemical and genetic analysis of dangerous pathogens without the need for full containment facilities.

The study is published in PNAS.

Study Finds Compounds that Boost Neurogenesis: Possible moa for Dimebon

Image: Flickr - be_khe
Following from positive phase II results, the announcement earlier this year that Dimebon (latrepirdine) failed to show a significant effect in a phase III clinical trial in Alzheimer’s patients was a major blow to patients, families and doctors. A study by researchers at UT Southwestern Medical Center has now shown that Dimebon can increase neurogenesis in adult rodent brains and have identified other, more potent compounds.

An in vivo screen of 1000 small molecules in adult mice identified eight compounds that were able to enhance neuron formation in the subgranular zone of the hippocampal dentate gyrus. One of the compounds, P7C3, was selected for further study on the basis of favourable ADME predictions. Daily administration of P7C3 to aged rats for 7 days was shown to enhance hippocampal neurogenesis relative to control animals and, after 2 months, treated rats performed significantly better in the Morris water maze test which provides a measure of learning and memory.
Dimebon and P7C3 structures
P7C3 exerts its proneurogenic effects by protecting newborn neurons from apoptosis and the team next compared the activity of P7C3 with that of Dimebon, which is also believed to have anti-apoptotic activity. Dimebon was found to be proneurogenic in vivo, albeit at levels 10-30 times higher than P7C3, raising the possibility that the two compounds may share a common mechanistic pathway. Although this idea can only be rigorously tested after identification of the molecular target(s), the study raises the hope that more potent analogues of Dimebon with improved clinical efficacy could be identified and also provides appropriate assays.

The study is published in the journal Cell.

Mild Stress Could Reduce Cancer

Image: Flickr - árticotropical
Whilst the effects of social and environmental factors on many aspects of health are relatively well understood, their influence on the progression of systemic cancer is much less well defined. A team of investigators led by scientists at The Ohio State University have now investigated the effect of an enriched environment – a more challenging setting that causes mild stress – on the growth of cancer in mice. The study found that an enriched environment – consisting of more complex housing, regular exposure to novel objects, and more exercise and social stimulation – led to a remarkable suppression of cancer proliferation in models of melanoma and colon cancer, even if delayed until the tumour was well established.

The researchers went on to identify the molecular pathways involved and discovered the enriched environment led to activation of a system known as the hypothalamic-sympathoneural-adipocyte (HSA) axis by brain-derived neurotrophic factor (BDNF). Activation of the HSA axis is proposed to increase sympathetic nervous system outflow to adipocytes, resulting ultimately in reduced secretion of leptin and increased secretion of adiponectin. The β-blocker, propanolol, was shown to inhibit the changes in circulating levels of leptin and adiponectin brought about by an enriched environment and also to block the inhibition of tumour growth, suggesting a link between β-adrenergic activity in white adipose tissue, circulating leptin/adiponectin levels and tumour growth.

The researchers propose that adipokines, released by white adipose tissue in response to hypothalamic BDNF-induced sympathetic outflow caused by mild stress, act as the major downstream effectors of a complex regulatory network leading to the antiproliferative phenotype. Direct gene transfer of BDNF was found to mimic the beneficial effects of an enriched environment, suggesting that either pharmacological or environmental induction of hypothalamic BDNF could slow the growth of tumours.

The study is published in the journal Cell.

Genetic Basis for Alopecia Areata

Alopecia Areata
Alopecia areata

Image: Wikimedia Commons

Alopecia areata is a type of hair loss that typically begins with one or more small bald patches on the scalp, beard area or elsewhere. The patches appear quite quickly and the hair may re-grow after a few months – or the condition may persist for several years with recurrences of patchy baldness and hair re-growth. The condition can also result in total loss of scalp hair (alopecia totalis) and, in a small number of cases, total loss of all body hair (alopecia universalis).

Alopecia areata is thought to be an autoimmune disease in which the immune system attacks the hair follicle, although the follicle is not destroyed since hair can re-grow. There also appears to be a hereditary component to the disease and a team lead by investigators at Columbia University Medical Center has now identified eight regions in the genome that are linked to the condition. The associated regions include some that have been linked to other autoimmune diseases including type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, celiac disease, and systemic sclerosis. Of particular interest for its potential role in the onset of disease is the ULBP (cytomegalovirus UL16-binding protein) gene cluster that has not previously been associated with autoimmune disease. Expression of ULBP3 proteins, which act as activating ligands for the NKG2D receptor on natural killer cells, is markedly upregulated in hair follicles affected by alopecia areata.

The study is published in the journal Nature.

Since drugs that target some of the pathways involved in alopecia areata have already been developed to treat other autoimmune diseases, the researchers hope that their discovery will lead quickly to treatments for hair loss caused by alopecia areata. The team are also developing a genetic test that should be able to help predict the likely course of the disease in a particular individual.