neurology – Page 3 – Drug Discovery Opinion

Glimepiride Potential for Treatment of Prion Diseases

Top - PrPc; Bottom - PrPSc Source: European Commission — Top - PrP^c; Bottom - PrP^Sc

Source: European Commission

Prion diseases comprise the transmissible spongiform encephalopathies, including scrapie in sheep, bovine spongiform encephalopathy (BSE, “Mad Cow” disease) in cattle and Creutzfeldt-Jakob disease in humans. Central to these diseases is the conversion of normal cellular prion protein (PrP^c) into the abnormally folded, pathogenic species (PrP^Sc) in the brain. The misfolding results in prion protein with distinct biochemical properties compared to the normal protein, such as reduced solubility and decreased susceptibility to proteases. Aggregates of PrP^Sc accumulate in association with neurons in affected brain areas, which is thought to lead to the synapse degeneration and neuronal death observed in infected hosts.

Researchers in the UK and Italy have now shown that glimepiride, a sulfonyl urea approved for the treatment of non insulin dependent diabetes mellitus (NIDDM), is able to reduce PrP^Sc formation in cell culture. The rationale for the study was based on the knowledge that generation of PrP^Sc is dependent on the presence of PrP^c and that this appeared to require PrPc expressed at the cell surface. PrP^c is linked to the membrane by a glycosylphosphatidylinositol (GPI) anchor and can be released from the surface of cells by treatment with phosphatidylinositol-phospholipase C (PI-PLC). Consistent with the hypothesis that cell-surface PrP^c is required, treatment of prion-infected neuronal cells with PI-PLC reduced PrP^Sc formation.

Since glimepiride has been shown to stimulate the release of some GPI-anchored proteins in adipocytes (via stimulation of an endogenous GPI-PLC), the team explored the effects of the drug on PrP^c/PrP^Sc in neuronal cell culture. Similarly to PI-PLC, glimepiride reduced the amount of cell-surface PrP^c in primary cortical neurons and neuronal cell lines. In addition, glimepiride reduced formation of PrP^Sc in three prion-infected neuronal cell lines.

The study, published in PLoSone, also demonstrated that glimepiride treated neurons were resistant to the toxicity of a PrP-derived peptide, PrP82-146.

The team note that modulation of cell-surface PrP^c may also have application in Alzheimer’s disease since it is a receptor for β-amyloid oligomers. Whether glimepiride is sufficiently CNS-penetrant to be effective remains to be seen.

Panic!

Tarantula psalmopoeus cambridgei - Image: Wikipedia — *Tarantula psalmopoeus cambridgei*

Image: Wikipedia

The body has no way to detect the absence of oxygen and feelings of suffocation are triggered instead by high levels of carbon dioxide. The ‘false-suffocation-alarm theory’ proposes that this alarm is triggered inappropriately in patients with panic disorder but, although several studies have linked high carbon dioxide levels with panic attacks in susceptible individuals, the reasons for this have not been clear. Researchers at the University of Iowa have now shown that inhaled carbon dioxide increases acidity in the brain and evokes fear by activating an acid-sensing ion channel (ASIC1a) in the amygdala. The amygdala plays a key role in the processing and memory of emotional reactions, including fear, but it was not known whether it also directly senses fear-evoking stimuli. ASICs are activated in vitro when extracellular pH falls, and although acidic pH modifies the activity of many receptors and proteins, few others are activated by extracellular acidosis, and few are as exquisitely pH sensitive as ASICs.

In tests in mice, increased carbon dioxide levels led to exaggerated innate and learned fear responses which could be blunted either by disrupting the ASIC1a gene or by pharmacological inhibition of ASIC1a using either the tarantula toxin, psalmotoxin, or A-317567.

The finding that ASIC1a channels in the amygdala act as chemosensors provides a molecular mechanism by which carbon dioxide can trigger fear and anxiety and suggests that targeting brain pH or ASIC channels could lead to new therapies for panic and anxiety disorders.

The study is published in the journal Cell.

Epilepsy Drugs as Neuroprotectants?

Calcium signalling plays a vital role in the survival of brain neurons and increased intracellular calcium has been identified as an early event triggering neuronal death in age-related neurodegenerative disorders such as Alzheimer’s disease. Additionally, L-type voltage-gated calcium channels have been implicated in neuronal death during aging.

Researchers at Washington University, Missouri, have now evaluated antagonists of both L-type and T-type calcium channels using an in vitro neuronal culture model. In the model, configured to monitor long- and short-term survival, nimodipine, an L-type calcium channel blocker originally developed as an anti-hypertensive, was neuroprotective in both assays. The anti-epileptic T-type calcium channel blocker, trimethadione, and mibefradil, an antihypertensive T- and L-type channel blocker, were neuroprotective in the short-term but not the long-term assay.

The results, published in the journal Molecular Degeneration, suggest that more than one calcium signalling pathway may be involved in regulating neuronal survival. Clinical evaluation of patients receiving calcium channel blockers may provide better insight into their benefit in terms of cognitive function in neurodegenerative disorders. The absence of effective treatments for age-related neurodegeneration should encourage further studies to determine whether these established drug classes could have additional utility.

IL-6 Stimulates Amyloid Plaque Clearance

Microglial cells (stained brown) Image: Wikimedia — Microglial cells (stained brown)

Image: Wikimedia

Amyloid plaques, which deposit around nerve cells, and neurofibrillary tangles, which build up inside the cells, are the primary hallmarks of Alzheimer’s disease. Many researchers believe that the plaques trigger a cascade of events leading to disease pathology but, although this hypothesis is supported by animal studies, it has not been conclusively proved in humans. Amyloid deposition has also been suggested to lead to neuroinflammation, creating a positive feedback loop resulting in further amyloid accumulation and chronic inflammation. Polymorphisms leading to increased levels of interleukin-6 (IL-6), a pro-inflammatory cytokine which activates microglial cells, have been linked to Alzheimer’s disease, adding weight to this hypothesis.

Scientists at the Mayo Clinic were carrying out experiments to demonstrate that activated microglia exacerbate neurodegeneration when they discovered, unexpectedly, that the microglia cleared the plaques from the brain. They had believed that the microglia would be unable to clear the plaques and that the resulting inflammation would worsen the disease. To examine the effect of IL-6 on amyloid processing and deposition, the team over-expressed murine IL-6 (mIL-6) in the brains of transgenic mice expressing mutated forms of the human amyloid precursor protein. Instead of creating a neurotoxic feedback loop that exacerbated amyloid pathology, IL-6 had no effect on amyloid processing and enhanced plaque clearance.

The study, which was published online on October 14^th in the FASEB Journal, is the first to examine in detail the effect of mIL-6 on amyloid deposition in vivo and suggests that the use of inflammatory mediators to manipulate the immune response could lead to new therapeutic approaches for the treatment of neurodegenerative diseases such as Alzheimer’s disease.

A study by researchers at the Universities of Florida and Frankfurt, published earlier this year in the journal Acta Neuropathologica, also addressed the role of microglia in Alzheimer’s disease. The study showed that microglia in amyloid-laden, degenerating regions of the brains of Alzheimer’s disease patients are not activated but, instead, are senescent and dystrophic. The team suggest that loss of microglia may contribute to neurodegeneration and that finding ways to keep microglia alive and healthy would be better than trying to inhibit their function with anti-inflammatory drugs.

Phosphorylation Modulates α-Synuclein Neurotoxicity

Immunohistochemistry for α-synuclein showing positive staining (brown) of an intraneural Lewy-body in the Substantia nigra in Parkinson disease

Image: Wikimedia - Marvin 101

α-Synuclein, expressed primarily in neural tissue, is normally an unstructured, soluble, protein. Under some circumstances, however, it has the ability to aggregate and form insoluble fibrils. A missense mutation in α-synuclein, A53T, was the first defined genetic lesion in familial Parkinson’s disease. Subsequently, α-synuclein was identified as a major component of Lewy bodies, even in the more common later-onset variant of Parkinson’s, which in most cases does not involve a clear family history of the disease. Lewy bodies are also seen in a group of related disorders, synucleinopathies, which may share important pathways of pathogenesis with Parkinson’s disease.

A new study from scientists at Brigham and Women’s Hospital and Harvard Medical School, Università di Padova and Massachusetts General Hospital has now established a role for phosphorylation in the control of α-synuclein neurotoxicity. The group had previously identified Ser¹²⁹ phosphorylation as a key event in α-synuclein neurotoxicity in a Drosophila model. Ser¹²⁹ phosphorylation conferred toxicity to α-synuclein without a substantial increase in the number of fibrillar deposits, suggesting that nonfibrillar species of α-synuclein may be neurotoxic.

The latest work shows that α-synuclein is also phosphorylated at Tyr¹²⁵ in transgenic Drosophila expressing wild-type human α-synuclein and that this tyrosine phosphorylation protects from α-synuclein neurotoxicity in a Drosophila model of Parkinson’s disease. Further, the team found that there was an age-related decrease in Tyr¹²⁵ phosphorylation in humans (as well as in the transgenic Drosophila) and that this phosphorylation was undetectable in the brains of patients who had dementia with Lewy bodies.

The study, published in the Journal of Clinical Investigation, suggests that the loss of protective tyrosine phosphorylation may predispose to clinically relevant α-synuclein neurotoxicity in human disease.

GSK-3 – Master Controller of Neural Stem Cells

GSK-3 (glycogen synthase kinase-3) is a serine-threonine kinase that consists of two family members in mammals, α and β, which share 98% sequence identity in their kinase domains. The β-isoform has recently been identified as a mediator of neurogenesis under the control of DISC1, a protein encoded by a gene implicated in schizophrenia susceptibility. GSK-3 has also been shown to be involved in regulation of embryonic stem cell self-renewal – GSK3 inhibitors help in maintenance of pluripotency.

A new study from researchers at the University of North Carolina at Chapel Hill School of Medicine has now shown that GSK-3 is a key regulator of neural stem cell proliferation and differentiation. Neural stem cells progress through different stages – neural epithelial cells, radial progenitor cells and intermediate neural precursors – and radial progenitor cells are particularly important because they are thought to provide the majority of the neurons of the developing brain and to give rise to all the cellular elements of the brain. The researchers used a conditional knockout in a mouse model, deleting both isoforms of GSK-3 during the radial progenitor phase of development. This resulted in locking the radial progenitor cells in a proliferative state, with no generation of mature neurons. The next step is to determine whether switching GSK-3 back on can stimulate differentiation, leading to an increased number of mature neurons. The researchers suggest that understanding the role of GSK-3 in neurogenesis could have implications for patients with neuropsychiatric conditions such as schizophrenia, depression and bipolar disorder.

The study is published in the journal Nature Neuroscience.

Protease Inhibitor Treats Rare Genetic Brain Disorder

Lissencephaly (literally “smooth brain”) describes a set of rare conditions in which the foetal brain does not develop normally beyond the third or fourth month of pregnancy and, instead of the usual folds and grooves, the cerebrum has a partially or completely smooth appearance. The severity and range of symptoms varies, but include mental retardation, failure to thrive, difficulty swallowing, seizures, and psychomotor problems. A number of factors are believed to cause lissencephaly including viral infection during pregnancy, an interrupted blood supply to the foetus, and genetic mutation. Loss of one copy of the gene LIS1 prevents migration of immature nerve cells from deep in the brain to the surface of the emerging cerebral cortex and US and Japanese researchers have now shown, in mice at least, that the results of this mutation can be reversed during pregnancy, leading to more normal offspring.

human brain In mice with only one copy of the LIS1 gene, the enzyme calpain reduces LIS1 protein levels to less than half of normal near the surface of cells, leading to abnormal brain development similar to that seen in human lissencephaly. Daily intraperitoneal injections of the small molecule calpain inhibitor, ALLN (N-Acetyl-Leu-Leu-Nle-CHO), to pregnant mice restored levels of LIS1 protein and resulted in offspring with more normal brains and no signs of mental retardation. Although the technique will not be easy to extend to humans, this study is the first successful attempt to use a protease inhibitor to reverse a severe brain defect caused by a partial deficiency in one key gene, and offers a proof-of-principle that the genetic equivalent to human lissencephaly can be effectively reversed during pregnancy to produce more normal offspring.

The authors hope that the approach could be extended to in utero treatments for other defects in which a protease plays a role in degrading an essential developmental protein.

The study was published online on September 6^th in the journal Nature Medicine.

Influenza Infection Could Lead to Neurological Complications Later in Life

Neurological complications such as encephalitis have been associated with influenza outbreaks since the middle ages but links with neurodegenerative diseases are more controversial. The association was strengthened after the 1918 Spanish influenza pandemic when some patients who developed von Economo’s encephalopathy – an atypical form of encephalitis – went on to display symptoms of Parkinson’s disease. Although a recent study showed that a reconstituted virus is not directly neurotropic, the engineered virus did strongly induce a variety of cytokines, some of which have been implicated in the pathophysiology of Parkinson’s disease.

Providing new evidence for an association between influenza infection and neurodegenerative diseases, researchers at St. Jude Children’s Research Hospital, have now shown that mice that survive infection with a virulent H5N1 strain of avian influenza are more likely to show changes in the brain associated with neurological disorders such as Parkinson’s disease and Alzheimer’s disease. Using an antibody to the influenza virus nucleoprotein, the team were able to track the progress of the virus into the CNS: 2-3 days after infection, the virus appeared in the peripheral nervous system; by day 3, the virus had invaded the brain stem and, by day 7, the virus was found in areas of the midbrain including the substantia nigra pars compacta (SNpc), and the mice now showed Parkinson’s disease-like symptoms such as tremor and movement problems.

Although after three weeks there was no evidence of the virus in the nervous systems of the surviving mice, inflammation in the brain triggered by the infection persisted for the entire three month course of the study. The Parkinson’s disease-like symptoms disappeared as the flu symptoms eased but, 60 days later, the mice had lost almost 20% of dopamine-producing neurones in the SNpc. α-Synuclein, a protein which forms insoluble plaques in the brains of Alzheimer’s disease and Parkinson’s disease patients, was also found to accumulate in H5N1-infected cells, including those in the midbrain where key dopamine-producing cells are located. The authors propose that a significant loss of dopaminergic neurons and long lasting immune response caused by influenza infection may worsen the effect of a second trigger, leading to increased risk of neurological disorders, such as Parkinson’s disease, later in life.

The H5N1 strain used in this study is so virulent that 61% of the 433 people who have been infected to date have died and, for the survivors, it is too early to say whether they will develop neurological problems. The influenza pandemic now engulfing the world is caused by an H1N1 strain rather than an H5N1 strain and, although the neurological threat posed by this virus is still being examined, early indications are that the H1N1 pandemic strain carries a low neurologic risk.

The study is published in the August 10^th early edition of the Proceedings of the National Academy of Sciences.

Itching is Not Just Toned-Down Pain

Most animals and people experience itching from time to time, but the unpleasant sensation is usually short-lived and can be relieved by scratching. Itching caused by insect bites or allergic reactions can be treated with anti-histamines but, for some people, chronic generalised itching associated with conditions including eczema, psoriasis, liver or kidney disease, HIV and certain cancers, as well as the use of some drugs, is resistant to anti-histamine treatment and can be a debilitating condition.

Many scientists have regarded itching as simply a toned down version of pain but researchers at Washington University School of Medicine have now shown that itch-specific neurons exist in mice. Two years ago, the team identified the first ‘itch gene’ in the spinal cord. They showed that mice lacking the gene for the gastrin-releasing peptide receptor (GRPR) scratched less than normal littermates when exposed to itch-inducing stimuli. The team have now shown that if they destroy GRPR-expressing neurons in the spinal cord using a GRPR ligand conjugated to a neurotoxin (bombesin-saporin), scratching was reduced by more than 80% and, in some cases, eliminated completely. The mice showed normal motor control and continued to respond normally to pain, suggesting that there is an itch-specific neuronal pathway in the spinal cord. This is the first behavioural evidence for itch-specific neurons and could eventually lead to novel treatments which alleviate chronic itching without affecting the pain response.

The study was published online on August 6^th in Science.

P2X7 Antagonist Improves Recovery after Spinal Injury

Acute crush injury to the spinal cord is immediately followed by secondary tissue damage, linked to massive release of ATP and activation of high-affinity P2X₇ receptors. Researchers at the University of Rochester Medical Center have previously shown that intraspinal injection of the P2X₇ antagonist, adenosine 5′-triphosphate-2′,3′-dialdehyde (OxATP), improves outcomes of spinal injury in rats, but intraspinal injection – together with cardiovascular toxicity – makes this treatment unattractive for human trauma victims. In a new study, the team have shown that outcomes in rats can also be improved by systemic treatment with a different P2X₇ antagonist, the Coomassie dye, Brilliant Blue G.

Writing in the journal PNAS, they show that iv administration of Brilliant Blue G (10 or 50 mg/kg) 15 minutes after injury, and for three consecutive days, protected spinal cord neurons from purinergic excitotoxicity and also reduced local inflammatory responses, resulting in reduced spinal cord anatomic damage and improved motor recovery. After 6 weeks, treated animals recovered sufficiently to walk with a limp whereas untreated animals did not walk again. Although it seems unlikely that Brilliant Blue G would efficiently cross the intact blood brain barrier, the dye was found to accumulate in the lesions in the injured animals.

There is currently no effective treatment to prevent secondary damage in patients with acute spinal cord injury and the team hope that their work will lead to safe practical treatments that could be administered soon after injury to improve outcomes for spinal injury victims.

Brilliant Blue G is a noncompetitive inhibitor of rat and human P2X₇ receptors with IC₅₀ values of 10 and 200 nM respectively.

brilliant blue g fdc blue 1 structures The team chose to use Brilliant Blue G for their experiments because they saw structural and functional similarities with a food additive, FD&C blue dye No 1 (E133), used in a variety of processed foods and generally considered to be safe. A number of groups have now designed selective P2X₇ antagonists – some of which have entered the clinic – and it would be interesting to see the effect of these newer compounds in the rat spinal injury model. Because of the differing affinities of antagonists for rat and human receptors, care will be needed in the choice of appropriate molecules for study, and in extrapolation of results from rodents to humans.