'Mad Cow' Proteins Successfully Detected In Blood

From University of Texas Medical Branch at Galveston

Researchers at the University of Texas Medical Branch at Galveston (UTMB) have found a way to detect in blood the malformed proteins that cause "mad cow disease," the first time such "prions" have been detected biochemically in blood.

The discovery, reported in an article scheduled to appear online in Nature Medicine Aug. 28, is expected to lead to a much more effective detection method for the infectious proteins responsible for brain-destroying disorders, such as bovine spongiform encephalopathy (BSE) in cattle and variant Creutzfeldt-Jakob disease (vCJD) in humans. The blood test would make it much easier to keep BSE-infected beef out of the human food supply, ensure that blood transfusions and organ transplants do not transmit vCJD, and give researchers their first chance to figure out how many people may be incubating the disease.

"The concentration of infectious prion protein in blood is far too small to be detected by the methods used to detect it in the brain, but we know it's still enough to spread the disease," said UTMB neurology professor Claudio Soto, senior author of the Nature Medicine paper. "The key to our success was developing a technique that would amplify the quantity of this protein more than 10 million-fold, raising it to a detectable level."

Soto and the paper's other authors, UTMB assistant professor of neurology Joaquin Castilla and research assistant Paula Saá, applied a method they call protein misfolding cyclic amplification (PMCA) to blood samples taken from 18 prion-infected hamsters that had developed clinical symptoms of prion disease. PMCA uses sound waves to vastly accelerate the process that prions use to convert normal proteins to misshapen infectious forms.

Successive rounds of PMCA led to the discovery of prions in the blood of 16 of the 18 infected hamsters. No prions were found in blood samples that were taken from 12 healthy control hamsters and subjected to the same treatment.

"Since the original publication of a paper on our PMCA technology, we've spent four years optimizing and automating this process to get to this point," Soto said. "The next step, which we're currently working on, will be detecting prions in the blood of animals before they develop clinical symptoms and applying the technology to human blood samples."

Tests for infectious prions in cattle and human blood are badly needed. Because current tests require post-slaughter brain tissue for analysis, in the United States only cattle already showing clinical symptoms of BSE (so-called "downer cows") are tested for the disorder. This is true even though vCJD potentially can be transmitted by animals not yet showing symptoms of the disease. (Only two cases of BSE have been found in American cows so far.) And although British BSE cases have been in decline since 1992, scientists believe the British BSE epidemic of the 1980s could have exposed millions of people in the UK and Europe to infectious prions. The extent of the vCJD epidemic is yet unknown. So far the disease has killed around 180 people worldwide, but numbers could reach thousands or even hundreds of thousands in the coming decades. Prions have also been shown to be transmissible through blood transfusions and organ transplants.

"Who knows what the real situation is in cattle in the United States? And with people, we could be sitting on a time bomb, because the incubation period of this disease in humans can be up to 40 years," Soto said. "That's why a blood test is so important. We need to know the extent of the problem, we need to make sure that beef and the human blood supply are safe, and we need early diagnosis so that when scientists develop a therapy we can intervene before clinical symptoms appear--by then, it's too late."

Study: Brain Structures Contribute To Asthma

From University of Wisconsin-Madison

The mere mention of a stressful word like "wheeze" can activate two brain regions in asthmatics during an attack, and this brain activity may be associated with more severe asthma symptoms, according to a study by University of Wisconsin-Madison researchers and collaborators.

The study, which appears in the Proceedings of the National Academy of Sciences (Online, August 29, 2005), reveals a functional link between emotion processing centers in the brain and certain physiological processes relevant to disease.

"While this study was small, it shows how important specific brain circuits can be in modulating inflammation," says Davidson, director of the affective neuroscience laboratory and the Waisman Laboratory for Functional Brain Imaging and Behavior. "The data suggest potential future targets for the development of drugs and behavioral interventions to control asthma and other stress-responsive disorders."

Previous studies and clinical evidence have shown that stress and emotional turmoil adversely affect people with inflammatory diseases like asthma. And signs of inflammation have been shown to affect the brain. But until now, nobody knew exactly what brain circuits were involved in these seemingly intertwined emotional and immune events or how the circuits might influence the severity of an acute asthma response.

Researchers used functional magnetic resonance imaging (fMRI) to scan the brains of six mildly asthmatic people who were asked to inhale ragweed or dust-mite extracts.

Subjects were then shown three types of words: asthma-related (such as "wheeze"), non-asthma negative (such as "loneliness") and neutral (such as "curtains"). Shortly after, researchers measured lung function in the subjects as well as molecular signs of inflammation in their sputum.

The fMRI scans revealed that the asthma-related terms stimulated robust responses in two brain regions--the anterior cingulate cortex and the insula--that were strongly correlated with measures of lung function and inflammation. The other types of words were not strongly associated with lung function or inflammation.

The two brain structures are involved in transmitting information about the physiological condition of the body, such as shortness of breath and pain levels, says Davidson, and they have strong connections with other brain structures essential in processing emotional information.

"In asthmatics, the anterior cingulate cortex and the insula may be hyper-responsive to emotional and physiological signals, like inflammation, which may in turn influence the severity of symptoms," says Davidson.
The researchers suspect that other brain regions may also be involved in the asthma-stress interaction.

Pinpointing The Cause Of A Neurodegenerative Disorder

From Howard Hughes Medical Institute

Researchers have discovered how the abnormal repetition of a genetic sequence can have disastrous consequences that lead to the death of neurons that govern balance and motor coordination. The studies bolster the emerging theory that neurodegenerative disorders can be caused by having extra copies of a normal protein, not just a mutated one.

People who are afflicted with the rare neurodegenerative disorder spinocerebellar ataxia type 1 (SCA1) suffer damage to cerebellar Purkinje cells caused by a toxic buildup of the protein Ataxin-1. Researchers knew that SCA1, Huntington's disease and other related disorders arise because of a “genetic stutter,” in which a mutation causes a particular gene sequence to repeat itself. These abnormal genetic repeats cause the resulting proteins to contain unusually long repetitive stretches of the amino acid glutamine.

The new findings, which are published in the August 26, 2005, issue of the journal Cell, provide a molecular explanation for Ataxin-1's assault on cerebellar Purkinje cells.

People with polyglutamine repeat disorders suffer severe degeneration in particular groups of neurons that vary depending on the type of disease. In SCA1, for example, the buildup of Ataxin-1 damages the cerebellar Purkinje cells. As a result of the damage, people with SCA1 lose balance and motor coordination. Loss of muscle control worsens until patients can no longer eat or breathe.

“We had known that the expansion of the glutamine tract within Ataxin-1 probably interfered with normal clearance of Ataxin-1, meaning that it accumulated in cells,” said Zoghbi. She noted that earlier studies yielded hints that the glutamine repeats somehow caused Ataxin-1 function to be altered in a way that damaged or killed Purkinje cells.
“We had been accumulating clues that the glutamine tract expansion is clearly what is important for disease because that's the mutation,” said Zoghbi. “But we also concluded that there was something else beyond the glutamine that's really mediating the toxicity of the protein.” Those conclusions were based, in part, on experiments in mice that showed that increased levels of normal Ataxin-1 can cause the pathology of SCA1.

Turning to the fruit fly, Drosophila, a favorite of geneticists, Zoghbi and her colleagues showed that a particular domain of Ataxin-1 was responsible for causing the flies to lose sensory neurons, but the domain's function remained unknown. Then, a finding by co-author Hugo Bellen, an HHMI investigator at Baylor, set the researchers off in a more fruitful direction. Bellen's team was doing experiments designed to identify proteins that interact with the Drosophila protein, Senseless. His group discovered serendipitously that Senseless interacts with the Ataxin-1 domain and is important for nervous system development.

In further experiments in flies, Zoghbi and her colleagues showed that increases in Ataxin-1 reduced levels of Senseless during peripheral nervous system development, causing developmental abnormalities. Additional experiments demonstrated that enhanced levels of normal and abnormal human Ataxin-1 produced even more serious pathology in the flies.

The researchers then showed that the same interaction and pathological effects occurred in mice — in which Ataxin-1 affected the mammalian version of Senseless, which is called GFi-1. And, they found that mice lacking GFi-1 showed Purkinje cell degeneration, just like humans with SCA1.

“The overall picture we have now is that glutamine expansion causes some aspects of the pathology of SCA1 in part by enhancing the activity of the domain that is outside the glutamine repeat,” said Zoghbi.

The finding offers insight into the molecular mechanisms that cause SCA1, Huntington's disease and other glutamine repeat disorders, said Zoghbi. “It seems to be a recurring theme in neurodegenerative disorders that having extra copies of a normal protein, not just a mutated one, can cause pathology. There have been observations that having extra copies of the normal alpha synuclein protein that causes Parkinson's disease, or of the amyloid precursor protein that causes Alzheimer's disease, can cause pathology,” she said. “So, this raises the question of whether mutations in the genes for these proteins enhance their normal action.

“Importantly, such insights can now guide studies that focus on the normal function and interactions of these proteins and how they might be enhanced by disease-causing mutations," said Zoghbi. "These studies could give better understanding of how the proteins cause disease.”

Loss of ALS2 Function Is Insufficient to Trigger Motor Neuron Degeneration in Knock-Out Mice But Predisposes Neurons to Oxidative Stress

From The Journal of Neuroscience, August 17, 2005, 25(33):7567-7574

Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease, is caused by a selective loss of motor neurons in the CNS. Mutations in the ALS2 gene have been linked to one form of autosomal recessive juvenile onset ALS (ALS2). To investigate the pathogenic mechanisms of ALS2, we generated ALS2 knock-out (ALS2-/-) mice. Although ALS2-/- mice lacked obvious developmental abnormalities, they exhibited age-dependent deficits in motor coordination and motor learning. Moreover, ALS2-/- mice showed a higher anxiety response in the open-field and elevated plus-maze tasks. Although they failed to recapitulate clinical or neuropathological phenotypes consistent with motor neuron disease by 20 months of age, ALS2-/- mice or primary cultured neurons derived from these mice were more susceptible to oxidative stress compared with wild-type controls. These observations suggest that loss of ALS2 function is insufficient to cause major motor deficits or motor neuron degeneration in a mouse model but predisposes neurons to oxidative stress.

New Target Found To Fight, Treat Parkinson's

From University at Buffalo

Neuroscientists from the University at Buffalo have described for the first time how rotenone, an environmental toxin linked specifically to Parkinson's disease, selectively destroys the neurons that produce dopamine, the neurotransmitter critical to body movement and muscle control.

Microtubules, intracellular highways that transport dopamine to the brain area that controls body movement, are the crucial target, they report.

Damage to microtubules prevents dopamine from reaching the brain's movement center, causing a back-up of the neurotransmitter in the transport system, the researchers found. The backed-up dopamine accumulates in the body of the neuron and breaks down, causing a release of toxic free radicals, which destroy the neuron.
The study appeared in the Aug. 9 issue of the Journal of Biological Chemistry.

"This study shows how an environmental toxin affects the survival of dopamine neurons by targeting microtubules that are critical for the survival of dopamine-producing neurons," said Jian Feng, Ph.D., assistant professor of physiology and biophysics in the UB School of Medicine and Biomedical Sciences and senior author on the study.
"Based on these findings, we have identified several ways to stabilize microtubules against the onslaught of rotenone. These results ultimately may lead to novel therapies for Parkinson's disease."

Feng and colleagues in the Department of Physiology and Biophysics have concentrated their research on the cellular mechanisms of the disease. They are interested specifically in understanding why rotenone destroys neurons that produce dopamine, while sparing neurons that produce other neurotransmitters.

Using cultures of rat neurons, the researches subjected neurons that produce various types of neurotransmitters to agents that mimic the action of rotenone. These results showed that dopaminergic neurons were destroyed while others survived.
They then topped off the treatment by adding the drug taxol, which stabilizes microtubules and prevents their breakdown. Findings showed that by protecting microtubules, the toxic effect of rotenone on dopamine-producing neurons was greatly reduced.

"Based on these findings, we believe that microtubules are a critical target of PD environmental toxins such as rotenone," said Feng. "Since many microtubule-depolymerizing agents are compounds naturally produced in many plants, our research points to the need to examine their possible link to Parkinson's disease. In addition, PD has a higher incidence in rural areas and is associated with pesticides and insecticides frequently used in farming practices."

The research also opens up novel avenues for the development of PD therapies by targeting microtubules, he said. Feng and colleagues in his laboratory are working actively towards this goal.

The Oral Antidiabetic Pioglitazone Protects from Neurodegeneration and ALS-Like Symptoms in Superoxide Dismutase-G93A Transgenic Mice

From The Journal of Neuroscience, August 24, 2005, 25(34):7805-7812

Amyotrophic lateral sclerosis (ALS) represents a fatal neurodegenerative disorder characterized by progressive death of the upper and lower motor neurons. Because accompanying inflammation may interact with and promote neurodegeneration, anti-inflammatory treatment strategies are being evaluated. Because peroxisome proliferator-activated receptor (PPAR) agonists act as potent anti-inflammatory drugs, we tested whether superoxide dismutase (SOD1)-G93A transgenic mice, a mouse model of ALS, benefit from oral treatment with the PPAR agonist pioglitazone (Pio). Pio-treated transgenic mice revealed improved muscle strength and body weight, exhibited a delayed disease onset, and survived significantly longer than nontreated SOD1-G93A mice. Quantification of motor neurons of the spinal cord at day 90 revealed complete neuroprotection by Pio, whereas nontreated SOD1-G93A mice had lost 30% of motor neurons. This was paralleled by preservation of the median fiber diameter of the quadriceps muscle, indicating not only morphological but also functional protection of motor neurons by Pio. Activated microglia were significantly reduced at sites of neurodegeneration in Pio-treated SOD1-G93A mice, as were the protein levels of cyclooxygenase 2 and inducible nitric oxide synthase. Interestingly, mRNA levels of the suppressor of cytokine signaling 1 and 3 genes were increased by Pio, whereas both the mRNA and protein levels of endogenous mouse SOD1 and of transgenic human SOD1 remained unaffected.

Identification of Aldolase as a Target Antigen in Alzheimer’s Disease

From The Journal of Immunology, 2005, 175: 3439-3445.

Alzheimer’s disease (AD) is the most common human neurodegenerative disease, leading to progressive cognitive decline and eventually death. The prevailing paradigm on the pathogenesis of AD is that abnormally folded proteins accumulate in specific brain areas and lead to neuronal loss via apoptosis. In recent years it has become evident that an inflammatory and possibly autoimmune component exists in AD. Moreover, recent data demonstrate that immunization with amyloid- peptide is therapeutically effective in AD. The nature of CNS Ags that are the target of immune attack in AD is unknown. To identify potential autoantigens in AD, we tested sera IgG Abs of AD patients in immunoblots against brain and other tissue lysates. We identified a 42-kDa band in brain lysates that was detected with >50% of 45 AD sera. The band was identified by mass spectrometry to be aldolase A. Western blotting with aldolase using patient sera demonstrated a band of identical size. The Ab reactivity was verified with ELISAs using aldolase. One of 25 elderly control patients and 3 of 30 multiple sclerosis patients showed similar reactivity (p < color="#000099">These findings reveal an autoimmune component in AD, point at aldolase as a common autoantigen in this disease, and suggest a new target for potential immune modulation.

Tissue Plasminogen Activator Promotes Matrix Metalloproteinase-9 Upregulation After Focal Cerebral Ischemia

From Stroke. 2005;36:1954.

Background and Purpose— Thrombolytic therapy with tissue plasminogen activator (tPA) in ischemic stroke is limited by increased risks of cerebral hemorrhage and brain injury. In part, these phenomena may be related to neurovascular proteolysis mediated by matrix metalloproteinases (MMPs). Here, we used a combination of pharmacological and genetic approaches to show that tPA promotes MMP-9 levels in stroke in vivo.

Methods— In the first experiment, spontaneously hypertensive rats were subjected to 3 hours of transient focal cerebral ischemia. The effects of tPA (10 mg/kg IV) on ischemic brain MMP-9 levels were assessed by zymography. In the second experiment, wild-type (WT) and tPA knockout mice were subjected to 2 hours of transient focal cerebral ischemia, and MMP-9 levels and brain edema during reperfusion were assessed. Phenotype rescue was performed by administering tPA to the tPA knockout mice.

Results— In the first experiment, exogenous tPA did not change infarct size but amplified MMP-9 levels in ischemic rat brain at 24 hours. Coinfusion of the plasmin inhibitor tranexamic acid (300 mg/kg) did not ameliorate this effect, suggesting that it was independent of plasmin. In the second experiment, ischemic MMP-9 levels, infarct size, and brain edema in tPA knockouts were significantly lower than WT mice. Administration of exogenous tPA (10 mg/kg IV) did not alter infarction but reinstated the ischemic MMP-9 response back up to WT levels and correspondingly worsened edema.

Conclusions— These data demonstrate that tPA upregulates brain MMP-9 levels in stroke in vivo, and suggest that combination therapies targeting MMPs may improve tPA therapy.

Translation elongation factor 1A is essential for regulation of the actin cytoskeleton and cell morphology

From Nature Structural and Molecular Biology, published online: 21 August 2005

The binding of eukaryotic translation elongation factor 1A (eEF1A) to actin is a noncanonical function that may link two distinct cellular processes, cytoskeleton organization and gene expression. Using the yeast Saccharomyces cerevisiae, we have established an in vivo assay that directly identifies specific regions and residues of eEF1A responsible for actin interactions and bundling. Using a unique genetic screen, we isolated a series of eEF1A mutants with reduced actin bundling activity. These mutations alter actin cytoskeleton organization but not translation, indicating that these are separate functions of eEF1A. This demonstrates for the first time a direct consequence of eEF1A on cytoskeletal organization in vivo and the physiological significance of this interaction.

A compensatory subpopulation of motor neurons in a mouse model of amyotrophic lateral sclerosis

From J. Comp. Neurol. 490:209-219, 2005.

Abstract
Amyotrophic lateral sclerosis is a fatal paralytic disease that targets motor neurons, leading to motor neuron death and widespread denervation atrophy of muscle. Previous electrophysiological data have shown that some motor axon branches attempt to compensate for loss of innervation, resulting in enlarged axonal arbors. Recent histological assays have shown that during the course of the disease some axonal branches die back. We thus asked whether the two types of behavior, die-back and compensatory growth, occur in different branches of single neurons or, alternatively, whether entire motor units are of one type or the other. We used high-resolution in vivo imaging in the G93A SOD1 mouse model, bred to express transgenic yellow fluorescent protein in all or subsets of motor neurons. Time-lapse imaging showed that degenerative axon branches are easily distinguished from those undergoing compensatory reinnervation, showing fragmentation of terminal branches but sparing of the more proximal axon. Reconstruction of entire motor units showed that some were abnormally large. Surprisingly, these large motor units contained few if any degenerating synapses. Some small motor units, however, no longer possessed any neuromuscular contacts at all, giving the appearance of winter trees. Thus, degenerative versus regenerative changes are largely confined to distinct populations of neurons within the same motor pool. Identification of factors that protect compensatory motor neurons from degenerative changes may provide new targets for therapeutic intervention.

An astrocytic basis of epilepsy

From Nature Medicine August 2005 - Vol 11 No 8

Hypersynchronous neuronal firing is a hallmark of epilepsy, but the mechanisms underlying simultaneous activation of multiple neurons remains unknown. Epileptic discharges are in part initiated by a local depolarization shift that drives groups of neurons into synchronous bursting. In an attempt to define the cellular basis for hypersynchronous bursting activity, we studied the occurrence of paroxysmal depolarization shifts after suppressing synaptic activity using tetrodotoxin (TTX) and voltage-gated Ca2+ channel blockers. Here we report that paroxysmal depolarization shifts can be initiated by release of glutamate from extrasynaptic sources or by photolysis of caged Ca2+ in astrocytes. Two-photon imaging of live exposed cortex showed that several antiepileptic agents, including valproate, gabapentin and phenytoin, reduced the ability of astrocytes to transmit Ca2+ signaling. Our results show an unanticipated key role for astrocytes in seizure activity. As such, these findings identify astrocytes as a proximal target for the treatment of epileptic disorders.

Nerve cell breakthrough is world first

SCIENTISTS in Edinburgh have created the world's first clutch of nerve stem cells in what could prove to be a major breakthrough in the race to treat diseases such as Parkinson's and Alzheimer's.
The cells were created in Edinburgh by the Institute for Stem Cell Research and the University of Milan. A team led by Professor Austin Smith developed the cells at the Edinburgh University-based institution.

It is a breakthrough because it is the first time that scientists have been able to grow and sustain pure brain cells. Until now, scientists had not been able to sustain the ability of neural stem cells to produce copies of themselves when grown in a dish. By changing the growth conditions for the cells, the Edinburgh and Milan labs have for the first time established pure stem cell divisions.

Researcher Steven Pollard said: "The purity of the cells, and the fact that they do not make tumours, means they should be valuable for studying the potential of transplantation to repair damage."
The long-term aim of the research is that the cells will be used to build replacement neural tissue for Alzheimer's and Parkinson's sufferers.

Alzheimer's disease is a progressive, irreversible brain disorder with no known cause or cure, and Parkinson's a disorder of the nervous system. The most likely immediate use for the artificially-created cells is to test out the effectiveness of new drugs. The scientists also hope that the cells will eventually help them to grow replacement brain tissue. The new technology could also lessen the need for animal testing.

Stem Cell Sciences plc (SCS) is the Edinburgh-based stem cell company which gained the licence to the new technology to derive and grow neural stem cells. Chief executive officer Dr Peter Mountford said: "Being able to grow pure brain cells is an exciting prospect for the company.

"SCS sees new business opportunities in both cell-based drug discovery and cell-based therapies for neurological disorders." The company's chief science officer, Dr Tim Allsopp, added: "The remarkable stability and purity of the cells is something unique in the field of tissue stem cells and a great step forward. "We have already had a number of approaches from pharmaceutical companies interested in using these cells to test and develop new drugs, and are looking forward to working with them to further develop and licence the technology."

Stem cells are "master" cells that can become many kinds of tissue, while nerve stem cells are those which help build the brain and central nervous system.

Worldwide research has been carried out on stem cells taken from adult tissue since the 1960's.

South Korean scientists stunned the medical world when they cloned 30 human embryos and developed them over several days last year.

Previous attempts at creating the nerve cells have produced contaminated samples that have not been scientifically useful.

And the breakthrough comes three months after scientists at Newcastle University announced they had successfully produced a cloned embryo using donated eggs and genetic material from stem cells.
It was the first time a human cloned embryo had been created in Britain.

Campaigners, including pro-life groups, have branded the research as "profoundly unethical" in the past. The creation of cloned babies is banned in the UK, but therapeutic cloning has been legal since 2002.

From Scotsman News

Mitochondrial changes in skeletal muscle in amyotrophic lateral sclerosis and other neurogenic atrophies

From Brain 2005 128(8):1870-1876.

Previous findings suggested specific mitochondrial dysfunction in skeletal muscle of patients with amyotrophic lateral sclerosis (ALS). To answer the question of whether the dysfunction is specific, we investigated the histochemical distribution of mitochondrial marker activities, the ratio of mitochondrial (mt) versus nuclear (n) DNA, and the activities of citrate synthase (CS) and respiratory chain enzymes in muscle biopsies of 24 patients with sporadic ALS. The data were compared with those in 23 patients with other neurogenic atrophies (NAs), and 21 healthy controls.

Muscle histology revealed similar signs of focally diminished mitochondrial oxidation activity in muscle fibres in both diseased groups. There was only minimal decline of mt/nDNA ratios in ALS and NA patients in comparison with healthy controls. The specific activities of mitochondrial markers CS and succinate dehydrogenase were significantly increased in both ALS and NA patients. The specific activities of respiratory chain enzymes were not significantly different in all three groups. It is concluded that the histochemical, biochemical and molecular mitochondrial changes in muscle are not specific for ALS, but accompany other NAs as well.

Mutant Cu/Zn Superoxide Dismutase (SOD1) Enzymes Implicated In Lou Gehrig's Disease

By American Society for Biochemistry and Molecular Biology, A new study indicates that mutant Cu/Zn superoxide dismutase (SOD1) enzymes that are associated with an inherited form of Lou Gehrig's disease cause the protein to become sticky in tissues. Partial unfolding of the mutant protein can expose hydrophobic residues that may promote abnormal interactions with other proteins or membranes in the cell.Over 5,600 people in the U.S. are diagnosed with amyotrophic lateral sclerosis (ALS) or Lou Gehrig's disease each year. About 30,000 Americans have the disease at any given time, and 10% of cases are inherited.

"Amyotrophic lateral sclerosis is a neurodegenerative disorder in which neurons of the motor pathways in the brain and spinal cord die," explains Dr. Lawrence J. Hayward of the University of Massachusetts Medical School. "It typically strikes during middle age, and although it may start with only mild weakness, the symptoms can spread insidiously over months to impair mobility, speech and swallowing, and ultimately the muscles required for respiration."

Despite the prevalence of ALS, the biological mechanisms that kill the motor neurons in most patients are incompletely understood. However, for a fraction of inherited ALS patients, mutations in the gene for SOD1 cause the disease by creating a toxic enzyme. Evidence suggests that misfolding or partial unfolding of mutant SOD1 proteins in these patients might be key to the toxicity.

Hoping to learn more about how SOD1 contributes to ALS, Dr. Hayward began to study the properties of several ALS-causing SOD1 mutants in research sponsored by the National Institutes of Health and the ALS Association. "Our efforts have focused upon trying to explain how over 100 different mutant forms of SOD1 cause inherited ALS," says Dr. Hayward. "The initial results were puzzling because some mutations had dramatic effects on copper and zinc binding, enzymatic activity, and stability of the protein, but many other mutations seemed to cause only subtle changes in these properties in vitro. Yet all of the mutants were known to be toxic in patients."

As a result of several additional experiments done in his lab and by other groups, Dr. Hayward suspected that the mutant proteins might be more vulnerable than the normal enzyme to specific stresses in tissues. In their Journal of Biological Chemistry paper, Dr. Hayward and his colleagues at the University of Massachusetts Medical School show that when the mutant SOD1 enzymes are exposed to reagents that can disrupt some of the protein's bonds or remove its metal ions, they become much stickier than the normal protein.

"The mutants, but not the normal SOD1, adhere to a hydrophobic or 'greasy' surface, and this property could promote abnormal interactions with other proteins or membranes in the cell," explains Dr. Hayward. "How well different tissues can handle this burden of sticky protein, especially during aging, may be one factor that determines which cell types are most vulnerable in the disease. It was interesting to us that the adherent forms were not restricted to the nervous system in the mouse models but were also seen in other tissues such as heart and skeletal muscle. It is possible that this property could contribute to abnormalities in muscle, while other tissues such as kidney do not accumulate hydrophobic SOD1 despite a high expression level of the mutants."

These results may lead to new treatments for some forms of ALS. For example, if researchers can minimize the hydrophobic exposure or can understand how certain tissues prevent build-up of the sticky forms of SOD1, they might be able to boost defenses in tissues known to be susceptible to mutant SOD1 accumulation.

Ref: J. Biol. Chem. 2005 280: 29771-29779.

Enzyme Action Creates Protein Linked To Alzheimer's Disease

From UT Southwestern Medical Center

Researchers at UT Southwestern Medical Center have defined a key step in the production of beta-amyloid, a short protein that is thought to be responsible for the development of Alzheimer's disease. Understanding this step may aid in the discovery of drugs that could help block the disease from developing.

In Alzheimer's disease, too much beta-amyloid is produced by an enzyme that has many other essential roles. As a result, simply blocking the whole enzyme knocks out many of its other functions - which is fatal to the organism.

Using cultured human and mouse cells, as well as test-tube assays, UT Southwestern researchers singled out how just one portion of the enzyme, a protein called nicastrin, is involved in the pathway that produces beta-amyloid, thereby leading to Alzheimer's disease. They hope next to work on ways to specifically block nicastrin. The study appears in the August 12 issue of the journal Cell. (Volume 122, Issue 3 , 12 August 2005, Pages 435-447 )

"The work provides an attractive potential strategy for developing treatment for Alzheimer's disease," said Dr. Gang Yu, assistant professor in the Center for Basic Neuroscience and of cell biology and senior author of the study. The research uncovered an "unprecedented mechanism of biochemistry," Dr. Yu said.

Nicastrin is a large protein that is a component of an enzyme called gamma-secretase, which is lodged in the cell's membrane. When it is at the cell surface, nicastrin sticks out into the area outside the cell. It has been thought to play a key role in the creation of a protein called amyloid-beta - the prime suspect for the damage Alzheimer's does to the brain - but the exact mechanism was unknown.

Dr. Yu and his colleagues found that nicastrin binds to several proteins lodged in the cell's membrane, including one called amyloid precursor protein, or APP. Nicastrin then guides membrane-bound proteins to the active area of gamma-secretase, which then splits the proteins. APP, for example, is chopped into two parts: amyloid-beta, which is then shipped to the outside of the cell, and another part that remains inside. Amyloid-beta forms the plaques seen in brains afflicted with Alzheimer's.

"Actually, it's quite a simple mechanism," Dr. Yu said. "Hopefully, we can screen for compounds that can block this process and find the exact pathways and how it can be regulated in Alzheimer's disease."

Now that nicastrin's function has been ascertained, it opens a way to block just the splitting of APP, leaving all the enzyme's other functions intact. For instance, it may be possible to generate chemical compounds that specifically prevent nicastrin from latching on to APP. If APP doesn't attach to nicastrin, APP remains intact and harmless. Meanwhile, nicastrin would be free to bind all the other essential proteins that it works on.
"We want to find a particular way to block the recognition of APP but not the others," Dr. Yu said.

Gene Loss Accelerates Aging

Researchers have discovered that the loss of a gene called p63 accelerates aging in mice. Similar versions of the gene are present in many organisms, including humans. Therefore, the p63 gene is likely to play a fundamental biological role in aging-related processes.

"To study how the p63 gene works, we devised a system for eliminating it from adult mouse tissues. What struck us right away was that these p63 deficient mice were aging prematurely," says Alea Mills of Cold Spring Harbor laboratory, who led the research.

Mice that are born without the p63 gene do not survive. Therefore, Mills had previously conducted extensive studies of mice that are born with only one copy of the gene. Still, these animals die at a young age. So to study p63 function in adults, Mills and her colleagues devised a sophisticated molecular genetic technique that enabled them to eliminate both copies of the gene from particular tissues--including skin and other multi-layered epithelial tissues--after the animals reached maturity.

The effects of premature aging observed in these p63 deficient mice were hair loss, reduced fitness and body weight, progressive curvature of the spine, and a shortened lifespan.

"Aging and cancer are two sides of the same coin. In one case, cells stop dividing and in the other, they can't stop dividing. We suspect that having the right amount of the p63 protein in the right cells at the right time creates a balance that enables organisms to live relatively cancer-free for a reasonably long time," says Mills, who adds that this is the first time the p63 gene has been implicated in aging.

"I first presented these results at a meeting in Tuscany. I don't want to sound flippant, but if you have to grow old somewhere, that's about as good a place as any to do it," says Mills.

The study is published in the September issue of the journal Genes & Development (advance online publication August 17). The other researchers involved in the study were Scott Lowe, Ying Wu, Xuecui Guo, and first author William Keyes of Cold Spring Harbor Laboratory, and Hannes Vogel of Stanford University.

Scientists for the first time have cloned a dog.

DENVER, Colorado (AP) -- Scientists for the first time have cloned a dog. But don't count on a better world populated by identical, well-behaved canines just yet.

That's because the dog duplicated by South Korea's cloning pioneer, Hwang Woo-suk, is an Afghan hound, a resplendent supermodel in a world of mutts, but ranked by dog trainers as the least companionable and most indifferent among the hundreds of canine breeds.

The experiment extends the remarkable string of laboratory successes by Hwang, but also re-ignites a fierce ethical and scientific debate about the rapidly advancing technology.
Last year, Hwang's team created the world's first cloned human embryos. In May, they created the first embryonic stem cells that genetically match injured or sick patients.

Researchers nicknamed their cloned pal Snuppy, which is shorthand for "Seoul National University puppy." One of the dog's co-creators, Gerald Schatten of the University of Pittsburgh School of Medicine, describes their creation, now 14 weeks old, as "a frisky, healthy, normal, rambunctious puppy."

Researchers congratulated the Korean team on improving techniques that might someday be medically useful. Others, including the cloner of Dolly the sheep, renewed their demand for a worldwide ban on human reproductive cloning.
"Successful cloning of an increasing number of species confirms the general impression that it would be possible to clone any mammalian species, including humans," said Ian Wilmut, a reproductive biologist at the University of Edinburgh, who produced Dolly nearly a decade ago.

The dog cloning team tried to distance its work from commercial cloning. "This is to advance stem cell science and medicine, not to make dogs by this unnatural method," Schatten said.

On scientific terms, the experiment's success was mixed. More than 1,000 cloned embryos were implanted into surrogate mothers and just three pregnancies resulted. That's a cloning efficiency rate lower than experiments with cloned cats and horses. Details appear in Thursday's issue of the journal Nature.

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The mRNA for EF1A Is Localized in Dendrites and Translated in Response to Treatments That Induce Long-Term Depression

The mRNA for Elongation Factor 1 Is Localized in Dendrites and Translated in Response to Treatments That Induce Long-Term Depression

From The Journal of Neuroscience, August 3, 2005, 25(31):7199-7209

There is increasing evidence that long-lasting forms of activity-dependent synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD), require local synthesis of proteins within dendrites. Identifying novel dendritic mRNAs and determining how their distribution and translation is regulated is a high priority. We demonstrate here that the mRNA for the elongation factor 1 (EF1) is present in vivo in the dendrites of neurons that exhibit LTP and LTD, and that its translation is locally regulated. The subcellular distribution of EF1 mRNA differs from any of the dendritic mRNAs that have been described previously. In the hippocampus, the mRNA is highly expressed in cell bodies and is also concentrated in the zone of termination of commissural/associational afferents in the inner molecular layer, suggesting that mRNA localization is in some way related to the distribution of different types of synapses. Nevertheless, the localization of EF1 mRNA is not altered by prolonged periods of synaptic activation that are sufficient to cause a dramatic redistribution of Arc mRNA. Local application of the metabotropic glutamate receptor agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) led to dramatic increases in immunostaining for EF1 protein in dendrites, and treatment of hippocampal slices with DHPG, which is known to induce LTD, led to increases in EF1 protein levels. Both responses were blocked by the protein synthesis inhibitor anisomycin. In contrast, stimulation of the perforant path using patterns of stimulation that induce LTP caused rapid increases of immunostaining for EF1 protein in the activated dendritic lamina, but these increases were not blocked by anisomycin or rapamycin. The findings suggest that local synthesis of EF1 protein may be important for the synaptic mechanisms that underlie protein synthesis-dependent LTD.

Early nuclear translocation of endonuclease G and subsequent DNA fragmentation after transient focal cerebral ischemia in mice

From Neuroscience Letters Volume 386, Issue 1 , 23 September 2005, Pages 23-27

Abstract
We investigated whether the endonuclease G (endoG) translocated from mitochondria to nucleus after transient focal cerebral ischemia (tFCI), thereby contributed to subsequent DNA fragmentation. Adult male mice were subjected to 60 min of focal cerebral ischemia by intraluminal suture blockade of the middle cerebral artery. Western blot analysis for endoG was performed at various time points of tFCI. Nuclear endoG was detected as early as 4 h after tFCI in the ischemic brain, and correspondingly mitochondrial endoG showed a significant reduction at 4 h after reperfusion (p <>. Immunohistochemistry of endoG confirmed that the nuclear translocation of endoG was detected as early as 4 h after tFCI in the middle cerebral artery (MCA) territory of the ischemic brain. Double immunofluorescent staining with endoG and AIF showed that endoG was predominantly colocalized with AIF at 24 h after tFCI. Double staining with endoG immunohistochemistry and TdT-mediated dUTP-biotin nick end labeling showed a spatial relationship between endoG expression and DNA fragmentation at 24 h after tFCI. These data suggest that the early nuclear translocation of endoG occurs and could induce DNA fragmentation in the ischemic brain after tFCI.

Gene Expression In The Aging Brain

From Public Library of Science

While medical science and a healthy lifestyle can help increase life expectancy, many aspects of aging and longevity are beyond our control. Published this week in the open-access journal PLoS Biology, Michael Eisen and colleagues shed light on these genetic factors by identifying sets of genes whose expression changes with age in human and chimp brains.

Using results from three microarray data studies performed in old and young humans and chimps in four different regions of the cortex (the brain region responsible for functions such as thinking), the authors found a pattern of age-related changes in the expression of hundreds of genes. Only one non-cortical region, the cerebellum (whose principal functions include movement), showed little change in age-related gene expression. Intriguingly, the set of affected cortical genes was different between humans and chimps. Since humans and chimps diverged about 5 million years ago - these changes have accumulated over a relatively short evolutionary time.

What might account for the difference? One common theory of aging holds that damage is done to DNA and proteins by "free-radicals" (highly reactive molecules produced by the metabolic activity of mitochondria). It predicts that more metabolically active tissues will show greater age-related differential gene expression. The results of this study support this theory since the more metabolically active cortex shows a greater reduction in gene activity.

These results raise questions about age-related gene expression changes, including whether metabolically active non-brain tissues display similar patterns, and whether the divergence between human and chimp patterns was the direct result of selection or an inevitable consequence of some other difference in brain evolution. The patterns seen here provide a starting point for understanding genetic changes in aging, and may reveal targets for treatment of neurodegenerative diseases.

Amphetamines Reverse Parkinson's Disease Symptoms In Mice

From Duke University Medical Center

Amphetamines, including the drug popularly known as Ecstasy, can reverse the symptoms of Parkinson's disease in mice with an acute form of the condition, according to new research at Duke University Medical Center. The team reports its findings in the August 2005 issue of Public Library of Science (PLoS) Biology.

The researchers caution that the findings in animals do not suggest Parkinson's disease patients should find relief by taking amphetamines, which are drugs of abuse with many dangerous side effects. The findings rather indicate that drugs with similar chemical attributes might offer useful alternatives to current therapies, the researchers said.

The new mouse model enables the researchers to acutely eliminate dopamine, exposing systems contributing to the disease that may not have been obvious before. In the current study, the researchers treated mice unable to recycle dopamine with a drug that also prevented them from manufacturing the brain messenger. The brains of the mice therefore lack detectable levels of dopamine and the animals exhibit all the symptoms of Parkinson's disease for up to 16 hours. Those symptoms included severely impaired movement, rigidity and tremor. When treated with L-DOPA, the symptoms disappeared as the animals resumed normal movement.

Surprisingly, the researchers reported, treating mice lacking dopamine with high doses of amphetamine derivatives – including methamphetamine and MDMA, otherwise known as Ecstasy – reversed those symptoms. Ecstasy was most effective at counteracting the manifestations of Parkinson's symptoms in the mice, with the beneficial effects becoming more pronounced with increasing dose.

The researchers also report that low doses of amphetamines could, when combined with L-DOPA, potentiate minimally effective doses of L-DOPA in the mice. This could have important considerations in reducing some of the side effects of current therapy.

"Taken together, the findings indicate that Ecstasy can improve movement control independently of dopamine and, most importantly provide evidence that drug activation of other neuronal pathways may be sufficient to restore movement even in the virtual absence of dopamine neurotransmission."

Using Nanoparticles To Repair Brain Cells Damaged By Disease, Trauma Or Stroke

Using Nanoparticles, In Vivo Gene Therapy Activates Brain Stem Cells: Technique May Allow Scientists To Repair Brain Cells Damaged By Disease, Trauma Or Stroke

From: University at Buffalo

Using customized nanoparticles that they developed, University at Buffalo scientists have for the first time delivered genes into the brains of living mice with an efficiency that is similar to, or better than, viral vectors and with no observable toxic effect, according to a paper published this week in Proceedings of the National Academy of Sciences.

In addition to delivering therapeutic genes to repair malfunctioning brain cells, the nanoparticles also provide promising models for studying the genetic mechanisms of brain disease.

Viral vectors can be produced only by specialists under rigidly controlled laboratory conditions. By contrast, the nanoparticles developed by the UB team can be synthesized easily in a matter of days by an experienced chemist.

The UB researchers make their nanoparticles from hybrid, organically modified silica (ORMOSIL), the structure and composition of which allow for the development of an extensive library of tailored nanoparticles to target gene therapies for different tissues and cell types. A key advantage of the UB team's nanoparticle is its surface functionality, which allows it to be targeted to specific cells.

This is the first time that a non-viral vector has demonstrated efficacy in vivo at levels comparable to a viral vector. In the future, this technology may make it possible to repair neurological damage caused by disease, trauma or stroke.

Journal: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0504926102

SCIENTISTS DISCOVER HOW NIPAH VIRUS ENTERS CELLS

From: National Institutes of Health

Working independently, two research teams funded by the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), have identified how Nipah and Hendra viruses, closely related viruses first identified in the mid-1990s, gain entry into human and animal cells.

Nipah and Hendra are emerging viruses that cause serious respiratory and neurological disease. People can get these deadly viruses from animals. Beginning in 1994, public health officials have recognized disease outbreaks in Malaysia, Singapore, Bangladesh and Australia. Both viruses use a protein essential to embryonic development to enter cells and begin their often-fatal attack, report researchers at the University of California, Los Angeles (UCLA) and the Uniformed Services University of the Health Sciences (USUHS) in Bethesda, Maryland.

The UCLA team, headed by Benhur Lee, M.D., describes its findings in a "Nature" paper posted online on July 6. The report by the USUHS researchers, led by Christopher Broder, Ph.D., is appearing online the week of July 4 in the "Proceedings of the National Academy of Sciences".

Using different methods, both teams identified a specific cell surface receptor, Ephrin-B2, as the doorway used by Nipah and Hendra viruses to get inside cells. This receptor is found on cells in the central nervous system and those lining blood vessels. It is crucial for the normal development of the nervous system and the growth of blood vessels in human and other animal embryos. Ephrin-B2 is found in humans, horses, pigs, bats and other mammals, which explains the unusually broad range of species susceptible to Nipah and Hendra virus infection.

The team narrowed the search for the Nipah/Hendra receptor by first sifting through the genetic sequences of 55,000 possible receptors using microarray technology as a molecular "sieve."

The scientists compared microarray signals from the 55,000 genetic sequences in one set of Nipah virus-resistant human cells with microarray signals from three sets of human cells that the virus can infect. This enabled the research team to narrow the possible number of receptor proteins to 120 by identifying those present in the virus-susceptible cells but absent in the virus-resistant cells. They winnowed the possibilities further -- to just 21 -- by selecting only those candidate receptors within the molecular weight range they expected. They selected 10 expressed at high levels in the susceptible cell lines and inserted them, one by one, into the cells that resisted Nipah virus to identify the receptor. When they inserted the gene for Ephrin-B2, the previously Nipah-resistant cells admitted the virus.

The UCLA team, with collaborators at the University of Pennsylvania, took a different approach, using tools of advanced molecular biology as well as old-fashioned detective work to identify the Ephrin-B2 receptor. They knew the receptor would be abundant among the type of cells Nipah virus attacks, that is, nerve cells and cells lining blood vessels.

To identify the human cell receptor, they created a bait: the Nipah protein that docks to that unknown receptor was attached to part of a human antibody, like a worm on a fishing hook. When they placed this bait onto cells susceptible to Nipah virus infection, it attached to a protein on the cell surface. When placed on Nipah-resistant cells, however, the antibody did not attach to the cells. The scientists used an instrument that sorts molecules by weight to identify that Ephrin-B2 was the cell receptor protein that bound to the antibody/Nipah protein "fishing pole" they had made.

They wanted to confirm their findings, but since they did not have access to a high-level biosafety laboratory as Dr. Broder's team did, the UCLA researchers engineered a harmless virus with Nipah virus proteins embedded in its coat. The UCLA team found that this artificial construct could infect cells vulnerable to Nipah virus but was unable to infect Nipah virus-resistant cells. They also showed that this engineered virus could infect nerve cells and cells lining blood vessels using Ephrin-B2 as the receptor, in the same way as actual Nipah virus would infect these cells.

Induction of endogenous neural precursors in mouse models of spinal cord injury and disease

From: European Journal of NeurologyVolume 12 Issue 8 Page 638 - August 2005

Adult neural precursor cells (NPCs) in the mammalian central nervous system (CNS) have been demonstrated to be responsive to conditions of injury and disease. Here we investigated the response of NPCs in mouse models of spinal cord disease [motor neuron disease (MND)] with and without sciatic nerve axotomy, and spinal cord injury (SCI). We found that neither axotomy, nor MND alone brought about a response by Nestin-positive NPCs. However, the combination of the two resulted in mobilization of NPCs in the spinal cord. We also found that there was an increase in the number of NPCs following SCI which was further enhanced by systemic administration of the neuregulatory cytokine, leukaemia inhibitory factor (LIF). NPCs were demonstrated to differentiate into astrocytes in axotomized MND mice. However, significant differentiation into the various neural cell phenotypes was not demonstrated at 1 or 2 weeks following SCI. These data suggest that factors inherent to injury mechanisms are required for induction of an NPC response in the mammalian spinal cord.

Mutant SOD1 alters the motor neuronal transcriptome: implications for familial ALS

From: Brain 2005 128(7):1686-1706

Abstract: Familial amyotrophic lateral sclerosis (FALS) is caused, in 20% of cases, by mutations in the Cu/Zn superoxide dismutase gene (SOD1). Although motor neuron injury occurs through a toxic gain of function, the precise mechanism(s) remains unclear. Using an established NSC34 cellular model for SOD1-associated FALS, we investigated the effects of mutant SOD1 specifically in cells modelling the vulnerable cell population, the motor neurons, without contamination from non-neuronal cells present in CNS. Using gene expression profiling, 268 transcripts were differentially expressed in the presence of mutant human G93A SOD1. Of these, 197 were decreased, demonstrating that the presence of mutant SOD1 leads to a marked degree of transcriptional repression. Amongst these were a group of antioxidant response element (ARE) genes encoding phase II detoxifying enzymes and antioxidant response proteins (so-called ‘programmed cell life’ genes), the expression of which is regulated by the transcription factor NRF2. We provide evidence that dysregulation of Nrf2 and the ARE, coupled with reduced pentose phosphate pathway activity and decreased generation of NADPH, represent significant and hitherto unrecognized components of the toxic gain of function of mutant SOD1. Other genes of interest significantly altered in the presence of mutant SOD1 include several previously implicated in neurodegeneration, as well as genes involved in protein degradation, the immune response, cell death/survival and the heat shock response. Preliminary studies on isolated motor neurons from SOD1-associated motor neuron disease cases suggest key genes are also differently expressed in the human disease.