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‘Resilience Factor’ Identified

bouncing ball — Image: Wikimedia Commons - Richard Bartz

Emotionally resilient people adapt to adversity and bounce back after stressful events; less resilient individuals find it difficult to cope with setbacks and may decline into depression or post-traumatic stress disorder (PTSD). Mice, like humans, vary widely in their reaction to stressful situations and a study led by researchers at Mount Sinai School of Medicine which was funded by the National Institute of Health’s National Institute of Mental Health (NIMH) has now uncovered a mechanism that helps to explain differences in resilience.

Mice experience stress when confronted by an aggressive, larger mouse and about two thirds of animals that repeatedly undergo this ‘social defeat’ have altered behaviours including long-lasting social avoidance and anxiety-like symptoms. The other third of the ‘defeated’ mice showed relatively few behavioural effects and these resilient animals were found to have higher levels of the transcription factor ΔFosB in the nucleus accumbens, an important brain reward-associated region or “pleasure centre”. Social behaviour in ‘defeated’ mice can be normalised by chronic antidepressant treatment and the action of fluoxetine was found to require induction of ΔFosB in the nucleus accumbens. Post-mortem examination shows that ΔFosB is also depleted in the brains of people who suffered from depression, suggesting that induction of this protein is a positive adaptation that provides resilience to stress.

ΔFosB is also known to be involved in regulating responses to both drugs of abuse and natural rewards such as food, sex and exercise, although the cell populations involved in these responses differ somewhat from those involved in protection from stress. The team suggest that concentrations of ΔFosB in the nucleus accumbens are important in setting the level of an individual’s reward-seeking motivation and that reduced concentrations of the protein are linked to the impaired motivation and ability to experience pleasure seen in many people with depression. The team now hope to discover small molecules that will augment the actions of ΔFosB and lead to resilience-boosting treatments for depression.

The study is published in the journal Nature Neuroscience.

Locking out or Locking in – It’s Not the Same Key for Malaria

Plasmodium parasites, responsible for malaria in humans, have a complex lifecycle that is dependent on mosquito and human hosts. In human blood, the merozoite stage of the parasite invades red blood cells (erythrocytes), growing and multiplying before rupturing the cell and escaping to infect other erythrocytes. It is this profound effect on erythrocytes that is responsible for the symptoms of malaria – fevers, chills and anaemia. Untreated, the disease can be fatal and drug resistance is an increasing problem. With up to half a billion people infected each year and nearly a million deaths, mostly in sub-Saharan Africa, there is an urgent need for new treatments.

Researchers at Harvard School of Public Health (HSPH) were attempting to identify the mechanism by which Plasmodium falciparum merozoites enter erthyrocytes, but instead found a parasite protein that is essential for escape from the cells. When the protein, P. falciparum calcium-dependent protein kinase (PfCDPK5), was suppressed the parasites were trapped in the host cell and unable to infect new cells. In further experiments the team showed that these merozoites were still able to invade erythrocytes if released from their host cell by other means, indicating separate mechanisms for invasion and egress from erythrocytes.

The findings reveal an essential step in the biology of P. falciparum and suggest a new, parasite-specific, drug target for fighting one of the world’s most common and dangerous infections. Whilst many scientists are looking for inhibitors of parasite egress and invasion of red blood cells, no anti-malarial drugs yet target these stages of the parasite lifecycle.

The study is published in Science.

Regulating Cholesterol Levels

Cholesterol is an essential component of all cellular membranes and is also required for synthesis of vitamin D and steroid hormones. Since it is poorly soluble in water, it is mainly transported through the bloodstream within lipoproteins – complex spherical particles composed of amphiphilic proteins and lipids whose outward-facing surfaces are water-soluble and inward-facing surfaces are lipid-soluble. Triglycerides and cholesterol esters are carried internally whilst phospholipids and cholesterol are transported in the surface monolayer of the lipoprotein particle. Several types of lipoproteins are found in blood, comprised of different apolipoproteins (which target specific tissues via receptor recognition) and with different capacities for cholesterol. These are usually referred to by their densities – the higher the ratio of cholesterol to lipoprotein, the lower the density. This gives rise to the so-called “bad cholesterol” (low density lipoprotein, LDL-cholesterol) and “good cholesterol” (HDL-cholesterol).

Problems arise when levels of the various lipoproteins are out of balance. Increased circulating levels of LDL-cholesterol are associated with the formation of foam cells, which can become trapped in the walls of blood vessels and contribute to artherosclerotic plaque formation leading to heart attacks and strokes. Conversely, HDL transports lipids to the liver for disposal and removes cholesterol from peripheral tissues, including the foam cells that form atherosclerotic plaques.

A new study by researchers at Massachusetts General Hospital (MGH) has now identified micro RNAs (miRNAs) that appear to play an important role in regulation of cholesterol/lipid levels. The team found that two members of the miR-33 family (miR-33a and miR-33b) target the ATP-binding cassette transporter A1 (ABCA1), an important regulator of HDL synthesis and reverse cholesterol transport, for posttranscriptional repression. Using antisense inhibition of miR-33 in mouse and human cell lines the researchers demonstrated up-regulation of ABCA1 expression and increased cholesterol efflux. Further, treatment of mice on a western-type diet with the antisense inhibitor resulted in elevated plasma HDL without affecting levels of LDL. The findings suggest that miR-33 may represent a therapeutic target for cardiovascular diseases.

The study is published in Science.

Newly Discovered Vulnerability in Tuberculosis

mycobacterium tuberculosis in sputum sample — Photomicrograph of a sputum sample containing *Mycobacterium tuberculosis* (stained red); Image - Wikipedia (source: CDC)

Tuberculosis (TB) is an airborne infectious disease caused by Mycobacterium tuberculosis (Mtb). TB is difficult to treat and the most commonly used antibiotics, rifampicin and isoniazid, need to be used for many months to eliminate the infection. The recent resurgence of TB, together with the emergence of drug-resistant strains of the bacterium, underscores the need for new treatments and researchers at Weill Cornell Medical College and the Novartis Institute for Tropical Diseases have identified a metabolic vulnerability in Mtb that could lead to new targets for drug therapy.

In vitro, Mtb is able to grow on a variety of carbon sources but fatty acids are believed to be the major source of carbon and energy for the bacterium during infection of a host. When bacterial metabolism is primarily fuelled by fatty acids, biosynthesis of sugars from intermediates of the tricarboxylic acid cycle is known to be essential for growth but the role of gluconeogenesis in the pathogenesis of Mtb had not been explored. Using genetic analyses and ¹³C carbon tracing, the team were able to show that phosphoenolpyruvate carboxykinase (PEPCK) – the enzyme that catalyses the first committed step of gluconeogenesis – is essential for the growth of Mtb supported by fatty acids. PEPCK was shown to be needed for growth of Mtb both in isolated macrophages derived from the bone marrow of mice and in infected mice.

Mtb lacking PEPCK failed to replicate in mouse lungs and silencing PEPCK during the chronic phase resulted in clearance of the infection, showing that Mtb relies on gluconeogenesis throughout the course of the infection. The finding that PEPCK plays a pivotal role in the growth and persistence of Mtb during both acute and latent infections in mice – and that PEPCK depletion also attenuates Mtb in IFNγ-deficient mice – suggests that this enzyme is an attractive target for chemotherapy.

The study is published in PNAS.

It May Not Be the Clock that’s Wrong

To ensure normal growth and avoid tumour formation, cell division must be tightly regulated. Remarkably, many cells from species as diverse as single celled organisms and humans only divide at certain times of the day, suggesting that division is under the control of a circadian clock. The evolutionary explanation put forward for this is a selective advantage for organisms with cells that divide at night when the mutational effects of ultraviolet light are lowest.

It has been proposed that the uncontrolled growth shown by tumour cells is caused by fault in the biological clock but a study by researchers at Vanderbilt University has now shown that although immortalised rat fibroblasts have functioning clocks, the clocks don’t control the rate at which the cells divide and grow. Similar results were seen in preliminary experiments with lung cancer cells. If follow-up studies confirm that control of cell cycle by circadian rhythm has been lost in immortalised cells, the research may suggest new targets for cancer therapy.

The study is published in PNAS.

Potent Inhibitor of Insulin Degrading Enzyme Reported

A new study from a Mayo Clinic-led research team has identified novel, potent inhibitors of insulin degrading enzyme (IDE). Despite an interest in IDE for over 50 years, because of its involvement in insulin catabolism, these are the first potent and selective inhibitors of the enzyme to be described. Given their peptidic nature the current compounds are unlikely to be drugs themselves, but the team hope that their findings will enable further exploration of IDE inhibition as a therapy for diabetes.

IDE is a ubiquitously expressed, secreted enzyme belonging to a small superfamily of zinc-metalloproteases that evolved independently of conventional zinc-metalloproteases. This difference is emphasised by the team’s finding that potent, non-selective hydroxamate inhibitors of zinc metalloproteases did not inhibit IDE.

Crystal structure of the Ii1-IDE complex (PDB ID 3E4A)

A high-throughput screening campaign failed to identify useful hits, so the researchers turned to a substrate-based approach leading to identification of Ii1 (Inhibitor of IDE 1), with a K_i of 1.7nM. Additional biostructural work identified the distinctive mechanism of IDE inhibition.

In vitro studies with the inhibitors, which included equipotent retro-inverso peptide analogues, demonstrated potent inhibition of extracellular insulin catabolism. In addition, and somewhat unexpectedly, IDE inhibition also enhanced insulin signalling, suggesting IDE involvement in intracellular degradation of insulin.

As well as cleaving insulin, IDE degrades a number of other substrates including atrial natriuretic peptide, glucagon and amyloid-β protein (Aβ). Indeed there has been considerable interest in up-regulating IDE activity as a potential therapy for Alzheimer’s disease (AD). The authors of the current study, published in PLoS ONE, suggest that any concern regarding negative impacts of IDE inhibition on AD could be addressed by developing inhibitors that do not cross the blood-brain barrier. Further, in light of the recent finding that intranasal insulin improves cognition in early AD patients, and given insulin’s beneficial effects on learning and memory, it may be overly simplistic to assume that IDE’s role in AD pathogenesis is limited to its predicted effects on Aβ alone.

Mice Show Pain, Just As We Do

mouse and man — Image: Flickr - bjormeansbear

We are used to the idea that facial expressions can tell us a lot about what our fellow humans are experiencing and researchers in Canada and the Netherlands have now found that the same is true for mice. In human volunteers, grimacing has been shown to match self-reported pain intensity and facial expression is also used to evaluate pain in patients who are unable to communicate orally.

Existing pain remedies do not work well for all patients and, in the search for more effective pain relief, mice and other rodents are used to develop new treatments. The present study has shown that mice, like humans, express pain through facial expressions and should provide a more accurate way to ensure that laboratory animals do not suffer unnecessarily and could also lead to better drugs for humans. The team have developed a ‘Mouse Grimace Scale’ by analysing high-resolution video images of mice before and during moderately painful stimuli – pain that is suggested to be comparable to a headache or that associated with an inflamed and swollen finger. The researchers, who are leaders in developing standards for facial expression to assess pain in human infants and others unable to communicate verbally, have proposed that five facial features in the mice should be scored. Several of these criteria – which are orbital tightening (eye closing), nose and cheek bulges, and ear and whisker positions – are similar to those used to assess pain in humans.

The study, which is the first to propose such a standardised and accurate scoring system linking facial expression to pain in animals, is published in the journal Nature Methods. Based on facial expression, trained researchers were able to correctly and reliably assess pain levels in real time. Previously, assessment of pain was limited to measuring withdrawal responses to pressure and heat and the ‘Mouse Grimace Scale’ may provide a more accurate way of predicting whether new treatments will eventually work in humans as well as reducing unnecessary pain in laboratory animals.

Link Between Sporadic and Familial ALS

Amyotrophic lateral sclerosis (ALS), or Lou Gehrig’s disease, is one of the most common neuromuscular diseases worldwide. The disease results in progressive loss of motor neurones leading to muscle weakening and atrophy, and is usually fatal within 5 years of the onset of symptoms. Most cases (90-95%) of ALS are sporadic with no evidence of inheritance but 5-10% of cases are familial and do have a hereditary component. Familial ALS has been linked to mutations in a number of genes including those encoding superoxide dismutase (SOD1) (~20% of familial cases), TAR DNA binding protein and, most recently, FUS (fused in sarcoma) protein (~5% of familial cases). The FUS protein is believed to be involved in DNA repair and transcription as well as RNA splicing and transport of RNA from the nucleus to the cytoplasm. Although normal FUS protein is largely confined to the nucleus, mutated FUS protein forms aggregates in the cytoplasm and these deposits have been correlated with degeneration of nerve cells.

Researchers from Northwestern University Feinberg School of Medicine have now shown that FUS protein forms characteristic skein-like cytoplasmic inclusions in spinal motor neurones in most cases of ALS, not just familial cases. Post-mortem examination of spinal cords and brains from 78 ALS sufferers and 22 controls showed that FUS pathology was present in all the ALS samples (except for those from patients with SOD1 mutations) but not in the control samples.

Although mutations in FUS account for only a small fraction of ALS, the study suggests that FUS protein may be a common component of the cellular inclusions in non-SOD1 ALS, whether familial or sporadic. Although the cause of ALS remains unknown, the identification of a common pathway in familial and sporadic ALS may spur the development of new cell-based and animal models of disease, and could eventually lead to new therapies for motor neurone diseases.

The study is published in the Annals of Neurology.

Another Activity for Vinpocetine

Researchers at University of Rochester Medical Center have now reported potent activity of vinpocetine in a mouse model of lung inflammation, showing promise for the treatment of chronic inflammatory diseases such as COPD, rheumatoid arthritis and psoriasis. The scientists have demonstrated that the mechanism of action is via inhibition of IκB kinase (IKK) resulting in suppression of the proinflammatory transcription factor, NFκB.

Since the compound has a long history of human use, the researchers hope that development of vinpocetine as an antiinflammatory therapy will be easier than for a novel molecule. The university has applied for a patent for vinpocetine for use as an IKK-inhibitor for the treatment of COPD and Yan and Berk, lead scientists of the study, have formed a start-up company, Rock Pharmaceuticals, with the hope of licensing the intellectual property rights.

Full details of the study are currently in press at PNAS, entitled “Vinpocetine inhibits inflammation via an IKK-dependent but PDE-independent mechanism”.

Liposomes can Deliver Wnt, Promote Bone Healing

bones — Image: Flickr - Anderson Mancini

Wnt signalling plays an important role in the development and maintenance of many organs and tissues, and appears to be especially important in regulating bone mass. Enhanced Wnt signalling has the potential to speed up healing and mice genetically modified to have prolonged Wnt signalling heal more quickly than control mice. So far it hasn’t been possible to directly test the effect of administering Wnt ligands because of difficulties in purifying and formulating the proteins, but researchers at Stanford University School of Medicine have now solved the problem by packaging the proteins in liposomal vesicles.

When Wnt3a-loaded liposomes were administered to mice with bone injury, within 3 days the animals had 3.5 times more new bone growth than animals that received no treatment or animals that received Wnt protein without the carrier liposomes. After 4 weeks, the bone had completely healed in the animals treated with Wnt-carrying liposomes whereas untreated animals took another 2 weeks. Wnt was shown to act by increasing the proliferation of bone progenitor cells – Wnt-responsive cells are found on the inner surface of the bone and participate in normal bone maintenance and in bone growth in response to injury. A liposomal formulation would also be suitable for use in people and could, after much further evaluation, be used to speed bone healing. Members of the bone morphogenetic protein family are currently used in spinal fusions and to treat some fractures but can cause bone to grow in the wrong place.

In some animals, such as flatworms and zebrafish, the Wnt pathway allows tissue regeneration without scarring and the authors hope that their study might also provide the basis for new treatments for stroke and heart attack where scar tissue formed as part of the normal healing process impairs later function.

The study is published in Science Translational Medicine.