neurology – Drug Discovery Opinion

Stressful ‘Lifestyle’ is Bad for Neurons

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

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.

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 GABA_A 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 GABA_A 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 GABA_A 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

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.

Targeting the Tangles

Although the relative importance of β-amyloid plaques and tau protein tangles in the progression of Alzheimer’s disease has been the subject of much debate, early emphasis was placed on the development of drugs to block production of β-amyloid. Although such compounds were shown to improve cognition in transgenic mice, unfortunately results from clinical trials have been more equivocal. Focus is now shifting to therapies that target tau pathology and, in a recent study, researchers from the University of Pennsylvania have identified a compound that reduced cognitive deficits in mutant human tau transgenic mice.

In healthy nerve cells, tau proteins interact with tubulin to stabilize axonal microtubules and promote tubulin assembly into microtubules. In Alzheimer’s disease and other ‘tauopathies’, hyperphosphorylated and misfolded tau proteins form insoluble neurofibrillary tangles that deplete levels of soluble tau and lead to destabilization of the microtubules and neuronal dysfunction. The team had previously proposed using microtubule-stabilising anti-cancer taxanes such as paclitaxel to treat tauopathies, but these do not penetrate the blood-brain barrier sufficiently well. The Penn team has now shown that once weekly treatment of tau transgenic mice with the brain-penetrant microtubule-stabilising agent, epothilone D, for three months significantly improved microtubule density and axonal integrity and also reduced cognitive deficits without notable side-effects.

The study, which is published in the Journal of Neuroscience, suggests that brain-penetrant microtubule-stabilising drugs could provide a new strategy for treating Alzheimer’s disease.

11β-HSD1 Inhibitor Improves Spatial Memory in Ageing Mice

Inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), a microsomal enzyme that converts cortisone into cortisol, are being developed to treat diabetes and metabolic disorders and now, in a study supported by the Wellcome Trust, researchers at the University of Edinburgh have shown that such compounds may also help to reduce – or even reverse – age-related memory loss. Such memory loss has been linked to increased activity of 11β-HSD1 and higher levels of glucocorticoids in the hippocamus, an area of the brain associated with memory.

Ageing mice display deficits in memory and learning similar to those experienced by some elderly people and life-long partial deficiency of 11β-HSD1 prevents this decline in transgenic mice. More surprisingly, improvements in memory – as judged by performance in a Y maze – were seen in mice after only ten days treatment with a selective 11β-HSD1 inhibitor, UE1961.

The team had previously shown that a non-selective 11β-HSD1 inhibitor, carbenoxolone, improves memory in healthy elderly men and in patients with type II diabetes after only one month of treatment. They now hope to complete preclinical assessment of the new compound and begin clinical trials within a year. The study is published in the Journal of Neuroscience.

Common Pathway for Neurodegenerative Diseases

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

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

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.

Study Finds Compounds that Boost Neurogenesis: Possible moa for Dimebon

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.

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.