Characterization of the caspase cascade in a cell culture model of SOD1-related familial amyotrophic lateral sclerosis

From Neuropathology & Applied NeurobiologyVolume 31 Issue 5 Page 467 - October 2005

There is increasing evidence that apoptosis or a similar programmed cell death pathway is the mechanism of cell death responsible for motor neurone degeneration in amyotrophic lateral sclerosis. Knowledge of the relative importance of different caspases in the cell death process is at present incomplete. In addition, there is little information on the critical point of the death pathway when the process of dying becomes irreversible. In this study, using the well-established NSC34 motor neurone-like cell line stably transfected with empty vector, normal or mutant human Cu-Zn superoxide dismutase (SOD1), we have characterized the activation of the caspase cascade in detail, revealing that the activation of caspases-9, -3 and -8 are important in motor neurone death and that the presence of mutant SOD1 causes increased activation of components of the apoptotic cascade under both basal culture conditions and following oxidative stress induced by serum withdrawal. Activation of the caspases identified in the cellular model has been confirmed in the G93A SOD1 transgenic mice. Furthermore, investigation of the effects of anti-apoptotic neuroprotective agents including specific caspase inhibitors, minocycline and nifedipine, have supported the importance of the mitochondrion-dependent apoptotic pathway in the death process and revealed that the upstream caspase cascade needs to be inhibited if useful neuro-protection is to be achieved.

Sgk1, a cell survival response in neurodegenerative diseases

From Molecular and Cellular Neuroscience Volume 30, Issue 2 , October 2005, Pages 249-264

Serum and glucocorticoid-regulated kinase 1 (sgk1) belongs to a family of serine/threonine kinases that is under acute transcriptional control by serum and glucocorticoids. An expanding set of receptors and cellular stress pathways has been shown to enhance sgk1 expression, which is implicated in the regulation of ion channel conductance, cell volume, cell cycle progression, and apoptosis. Recent evidence for the involvement of sgk1 in the early pathogenesis of MPTP-induced Parkinson's disease (PD) prompted us to investigate in more detail its expression and role in animal models of different neurodegenerative diseases.

Here, we show that transcription of sgk1 is increased in several animal models of PD and a transgenic model of amyotrophic lateral sclerosis (ALS). The upregulation of sgk1 strongly correlates with the occurrence of cell death. Furthermore, we provide evidence that the Forkhead transcription factor FKHRL1 and some of the voltage-gated potassium channels are physiological substrates of sgk1 in vivo. Using a small interfering RNA approach to silence sgk1 transcripts in vitro, we give evidence that sgk1 exerts a protective role in oxidative stress situations.

These findings underline a key role for sgk1 in the molecular pathway of cell death, in which sgk1 seems to exert a protective role.

Clues To Parkinson's, Huntington's, And ALS May Be Found In The Walking Patterns Of Affected Mice

From Mouse Specifics, Inc. (MSI)

In a published report in the Journal of NeuroEngineering and Rehabilitation, scientists from Harvard Medical School and Mouse Specifics, Inc. (MSI) in Boston have characterized gait dynamics in mice with Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS). Gait analysis is the process of quantification and interpretation of locomotion. In humans, gait analysis is widely used to quantify patients' movement disorders to provide diagnosis and treatment options. The distinct gait patterns of mice with Parkinson's, Huntington's, and ALS reflect impairment of specific neural pathways involved in the different aspects of the diseases, and provide the basis for testing new therapies.

Gait disturbances are characteristic of patients with Parkinson's disease, Huntington's disease, and ALS. Sudden falls due to unsteady gait are major hazards for the affected patients. Mouse models of these human diseases are essential to their understanding and treatment, yet gait disturbances in diseased mice are seldom described. The scientists from Harvard and MSI used a high speed digital imaging system and a recently patented treadmill with a transparent belt (The DigiGait™ Imaging System) to quantify gait indices in mice treated with toxins to mimic symptoms of either Parkinson's or Huntington's disease, and in mice genetically modified to replicate symptoms of ALS. As in patients, mice with Parkinson's and Huntington's disease exhibited less steady gait than healthy mice, with greater stride-to-stride variability of gait cycle timing. As in patients, upper limb dynamics were more variable in mice with Huntington's disease than in mice with Parkinson's disease. As in patients, gait variability was less disturbed in mice with ALS than in mice with Parkinson's. The altered gait dynamics in the different mouse models indicate that specific neural pathways are involved in the different observed gait pathologies.

"The ability to quantify and distinguish measures of 'walking' in mice with, say, Parkinson's disease and mice with Huntington's disease provides a new opportunity to zero in on the mechanisms underlying their distinct pathologies and characteristics," said Visiting Professor Ivo Amende, Medical University Hannover, Germany, lead author of the study. "Our hope is that gait analysis in mouse models of human movement disorders and neurodegenerative diseases will accelerate the development of drugs to prevent or reverse gait disturbances." The publication can be accessed via the Journal of NeuroEngineering and Rehabilitation website at http://www.jneuroengrehab.com/content/2/1/20 .

Molecule protects against developing Alzheimer's Disease

A molecule expressed by nerve cells may protect humans from developing Alzheimer's Disease (AD). In particular, it may reduce the risk of the formation of senile plaques in the brains of patients with AD, as researchers from the Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch (Germany) and their collaborators in Denmark, Australia, and the USA have been able to demonstrate. The findings by Dr. Olav M. Andersen, Professor Thomas Willnow (both from the MDC) and Dr. Anders Nykjær (University of Aarhus, Denmark) have been published online in PNAS* (doi:10.1073).

A hallmark of Alzheimer disease are protein plaques in the brain which accumulate over many years. They are derived from the amyloid precursor protein (APP) which for unknown reasons is chopped up into smaller fragments, including the amyloid beta peptide, which forms these dangerous plaques. The plaques destroy the patients' nerve cells and lead to dementia, impairing the patients memory, thinking, and behaviour. According to the National Institutes of Health (NIH) more than four million Americans suffer from AD, an incurable disease.

The older one gets, the greater the risk of developing this disease. It is estimated that about half of the individuals over 85 years of age are affected. Professor Willnow and his colleagues were able to demonstrate that the molecule, named sorLa (abb. sorting protein-related receptor), binds to APP in nerve cells and thus prevents its dissection into the amyloid beta peptide. They could also show that genetically modified mice which cannot produce sorLA have increased levels of amyloid beta peptides because APP is destructed at a much higher rate than in healthy animals.

The researchers also looked at the brains of patients who died from AD and compared them with subjects who had not suffered from this disease. Surprisingly, the nerve cells of the AD patients had not produced sorLA, but the nerve cells of the control group had done so. The researchers conclude that in individuals whose brains produce little or no sorLA, the uncontrolled production of amyloid beta peptides likely accelerates onset and progression of neurodegenerative processes, making sorLA an important risk factor for AD.

Ongoing research is directed towards identification of substances that could increase the production of sorLA in the brain of those people that produce insufficient amounts of the molecule. The researchers hope that in the future it will be possible to pharmacologically reduce the formation of these dangerous plaques in the brain by modulating sorLA levels.

The most infectious prion protein particles

From Nature Volume 437 Number 7056 pp169-294

The prospects of limiting the spread of transmissible spongiform encephalopathies such as Creutzfeldt−Jakob disease depend in part on identifying the most infectious forms of the prions that carry the diseases. A study of modified scrapie prions shows that clusters of 14 to 28 prion proteins are the most infectious and that clusters of less than six molecules have virtually no infectivity. That could have implications for the treatment of diseases such as Alzheimer's and Parkinson's, characterized by deposition of prion-related amyloid fibrils. It's possible that efforts to alleviate symptoms by destabilizing these large protein aggregates might make things worse by producing smaller, more infective particles. Two other papers in this issue tackle fundamental aspects of the biology of prions and amyloid fibrils. The conversion of the yeast protein Sup35 to its prion form does not need to happen during the synthesis of Sup35 — mature and fully functional molecules can readily join a prion seed. This remodelling of the mature protein is accompanied by the immediate loss of its activity. And a study of a 'designed' amyloid fibril made from ribonuclease A reveals that amyloid containing native-like molecules can retain enzyme activity. This involves a domain swap with the neighbouring protein, and supports the 'zipper-spine model' for -amyloid structures.

Montreal Researchers Probe The Genetic Basis Of Memory

From University of Montreal

A group of Montreal researchers has discovered that GCN2, a protein in cells that inhibits the conversion of new information into long-term memory, may be a master regulator of the switch from short-term to long-term memory. Their paper Translational control of hippocampal synaptic plasticity and memory by the eIF2a kinase GCN2, which was published in the August 25th issue of the journal Nature, provides the first genetic evidence that protein synthesis is critical for the regulation of memory formation.

"Not all new information we acquire is stored as long-term memory," says Dr. Costa-Mattioli, a post-doctoral fellow in the laboratory of Dr. Sonenberg, who spearheaded the research project. "For example, it takes most people a number of attempts to learn new things, such as memorizing a passage from a book. The first few times we may initially succeed in memorizing the passage, but the memory may not be stored completely in the brain and we will have to study the passage again."

In a series of experiments, the researchers demonstrated that mice bred without the GCN2 protein acquire new information that does not fade as easily as that of normal mice. This new information is more frequently converted into long-term memory. The researchers concluded that GCN2 may prevent new information from being stored in long-term memory.

Adds Dr.Jean-Claude Lacaille: "The process of switching to long-term memory in the brain requires both the activation of molecules that facilitate memory storage, and the silencing of proteins such as GCN2 that inhibit memory storage."

Although research on humans is still a distant possibility, the scientists believe their discovery may hold promise in the treatment of a variety of illnesses linked to memory. "The discovery of the role of GCN2 in long-term memory may help us develop targeted drugs designed to enhance memory in patients with memory loss due to illnesses such as Alzheimer's disease, where protein synthesis and memory are impaired," concludes Dr. Karim Nader

New Techniques Study The Brain's Chemistry, Neuron By Neuron

From University of Illinois at Urbana-Champaign

Researchers at the University of Illinois at Urbana-Champaign have developed tools for studying the chemistry of the brain, neuron by neuron. The analytical techniques can probe the spatial and temporal distribution of biologically important molecules, such as vitamin E, and explore the chemical messengers behind thought, memory and emotion.

"In most organ tissues of the body, adjacent cells do not have significant differences in their chemical contents," said Jonathan Sweedler, a William H. and Janet Lycan Professor of Chemistry and director of the Biotechnology Center at the U. of I. "In the brain, however, chemical differences between neurons are critical for their operation, and the connections between cells are crucial for encoding information or controlling functions."

By dismantling a slice of brain tissue into millions of single cell-size pieces, each of which can be interrogated by mass spectrometric imaging techniques, Sweedler's research group can perform cellular profiling, examine intercellular signaling, map the distribution of new neuropeptides, and follow the release of chemicals in an activity-dependent manner.

Sweedler will describe the techniques and present new results at the 230th American Chemical Society national meeting in Washington, D.C. Using these techniques, Sweedler's group has already discovered multiple novel neuropeptides in a range of neuronal models from mollusks to mammals.

"We work with sea slugs, whose simple brains contain 10,000 neurons; we work with insects possessing one million neurons; and we work with mice having 100 million neurons," said Sweedler, who also is a researcher at the Beckman Institute for Advanced Science and Technology. "Working with these model organisms allows us to examine the functioning of such basic operations as the neuronal control of behavior and long-term memory."
Sweedler's group also developed an approach for looking at the distribution of smaller molecules in brain cells. In a paper accepted for publication in the Journal of the American Chemical Society, and posted on its Web site, they report the subcellular imaging of vitamin E in the sea slug Aplysia californica.

The researchers utilized novel sampling protocols and single cell time-of-flight secondary ion mass spectrometry to identify and map the presence of vitamin E in the membranes of isolated neurons.
"To our surprise, we found that vitamin E was not distributed uniformly in the neuronal membrane," Sweedler said. "Instead, vitamin E was concentrated in the neuron right where it extends to connect with other neurons."
The subcellular localization of vitamin E, which had been impossible to obtain in the past, supports other work that suggested vitamin E performed an active role in transport mechanisms and cellular signaling of neurons.

"Our technique doesn't tell us how or why vitamin E is distributed this way, but suggests that it is under active control and tight regulation," Sweedler said. "Understanding the chemistry that takes place within and between neurons, including small molecules like vitamin E, will no doubt lead to a better understanding of brain function in healthy and diseased brains."

Virginia Tech Engineer Investigates Enzyme Link To Neurological Disease

From Virginia Tech

Several neurologically based afflictions, such as Huntington's, Parkinson's, and Alzheimer diseases, have been correlated to a higher than normal presence of a specific type of enzymes, called transglutaminases (TGase) in the human body. TGases, whose function is to catalyze covalent bonds among proteins, are commonly found in several different human tissues.

In the presence of unusually high levels of these enzymes, some proteins tend to form denser clusters than normal in vivo. If the aggregates grow in size, it can lead to a build-up of insoluble plaques that can block neurovascular transport and cause neural cell death.

"If higher TGase concentrations in cerebrospinal fluid and in the brain lead to protein agglomeration, then their inhibition could reduce symptoms, delay the onset of agglomeration, and maintain viable neural cell health extending the quality of life for those afflicted," hypothesizes Brian Love, a professor of materials science and engineering (MSE) at Virginia Tech.

Love, who focuses his research on tissue and cell engineering, and Elena Fernandez Burguera, a post-doctoral research associate, are evaluating specific therapies to fight the abnormally high TGase binding. Based upon the prior work of several others who are conducting clinical trials, Love and Burguera are developing an in vitro model to evaluate the ability of several inhibitors to block protein aggregation by TGases.

Again, based on the work of other scientists, "several compounds show some positive effects," Love says. These include creatine, cystamine hydrochloride, and a few others. "The development of an inhibition protocol may help test the efficacy of other inhibitors as well," the engineer adds.

The Virginia Tech researchers are looking at the enzymatic binding of protein-bound polystyrene particles as models. Groups of particles are dispersed in calcium-rich aqueous solutions containing TGases. Once mixed, the particle binding begins. The bigger agglomerates attempt to settle out of the solution, and Love and Burguera track particle sedimentation.

The tracking method, called Z-axis Translating Laser Light Scattering (ZATLLS), is unique to Virginia Tech and based on key concepts in transport phenomena. It has been used to gauge how other complex fluids, such as paints and sealants, are dispersed. Now Love and Burguera are resolving when protein coated particles are effectively dispersed in vitro and under what conditions they are unstable enough to agglomerate.

They track in situ sedimentation of protein-coated particles exposed to transglutaminase, both in the presence of and without transglutaminase inhibitors. "We can use ZATLLS to resolve whether inhibitors prevent agglomeration of protein coated particles by TGase if the inhibitors lower the particle sedimentation velocity,"

Love says. "Our goal is to find the safest and most effective inhibitors that prevent the agglomeration-based crosslinking found throughout these neurological disorders."

Brain Remembers Familiar Faces When Choosing Potential Mate

From University of Liverpool

Scientists at the University of Liverpool have discovered that the human brain favours familiar-looking faces when choosing a potential partner.

The research team found that people find familiar faces more attractive than unfamiliar ones. They also found that the human brain holds separate images of both male and female faces and reacts to them differently depending on how familiar it is with their facial features.

Dr Anthony Little, from the University's School of Biological Sciences, examined whether early visual experience of male and female faces affected later preferences. The research team asked over 200 participants to view a number of human faces that had been digitally manipulated to change their facial characteristics.

Dr Little said: "We found that participants preferred the face that they were most visually familiar with. In one of the tests we showed participants a block of faces with wide-spaced eyes and then asked them to compare these with a face that had narrow-spaced eyes. We found that participants preferred the face with wide-spaced eyes, suggesting that the brain connects familiarity with attraction."

The team also asked participants to judge the same preferred facial features in those of the opposite sex. Participants who were shown male faces with wide-spaced eyes preferred this trait in subsequent male faces but not in female faces.

Dr Little explains: "The research revealed that the sex of the face can be a deciding factor in facial preference. The tests showed for the first time that the brain holds separate visual patterns of male and female faces and responds to them based on their sex as well as their familiarity. We will continue to investigate why this is the case."

"The next step in the research is to find out why the brain makes a link between familiarity and attractiveness. It maybe that visual experience of particular facial features suggests that a person is 'safe' or more 'approachable', both of which are desirable traits."