DOI: 10.1126/science.opms.p0600009

Life Science Technologies
Aging and Neuroscience
Probing the Brain

Researchers face huge challenges as they seek to discover the causes and mechanisms of age-related neurological ailments such as Alzheimer's and Parkinson's diseases, and to translate their findings into effective methods of diagnosis and treatment. But emerging tools and technologies have begun to unravel some of the conditions' complexities.

By Peter Gwynne and Gary Heebner

Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, or by interviewee Marcelle Morrison-Bogorad or the National Institute on Aging. Nor is it meant to imply that their products or services are superior to those of other companies.

Neurological ailments that afflict patients as they age, such as Alzheimer's disease and Parkinson's disease, present medical scientists with one of their greatest challenges. While research continually increases knowledge of aging and related diseases, many of the details of how the brain works (and doesn't work) remain a mystery.

However, researchers have begun to make significant advances in recent years. Those include the discovery that new nerve cells can be made in the brain and spinal cord, the identification of biomarkers that can help to detect Alzheimer's disease before clinical diagnosis and signs of memory loss appear, and the finding that a mutation in a recently discovered gene (LRRK2) is the most common genetic cause of Parkinson's disease.

Plainly, studies of neurological diseases related to the aging process have begun to gather momentum. "The field is really fermenting these days," says Marcelle Morrison-Bogorad, director of the neuroscience and neuropsychology of aging program at the U.S. National Institute on Aging. "We are trying to accelerate the process of moving from basic research on pathways to translational science and taking the next step of deciding what compounds are at a stage when we can put them into clinical trials." Scientists who attended July's International Conference on Alzheimer's Disease and Related Disorders in Madrid, Spain, expressed similar optimism. "A lot of people said that this is the beginning of an important time for Alzheimer's," recalls Ron Black, senior director of neuroscience medical research at Wyeth.

Tools Old and New

To fulfill that promise, researchers rely on a variety of tools and technologies—both traditional and new. They include imaging and microscopy, cell culture, antibodies, and sophisticated assays. "Progress in genomics will continue to make some gains. Genomics has helped to pinpoint patterns of mutations associated with disease states and the heritability of certain diseases," says Daniel Tusé, vice president of business development for Predictive Diagnostics, a wholly owned subsidiary of Large Scale Biology Corporation. "Also important are diagnostic or medical imaging with MRI and CT scans that can suggest clinical interventions. And proteomics has a strong future. It will provide valuable snapshots if relevant biomarkers can be developed."

Christian Kier, marketing manager (confocal laser scanning microscopy) for Leica Microsystems, points out two key characteristics of today's tools. "Tools are coming into the mainstream, changing from expert user tools to instruments that everyone must use to play this game," he says. "Also, they have broadened in scope. They are now being used as analytical tools."

Current studies of neurological disease provide plenty of reason for hope in tackling neurological ailments. "Recent advances have made it easier to start on drug discovery," says David Jackson, research area manager for molecular and cellular biology at Invitrogen. "An evolving consensus has emerged over the past five years about what cell types participate in neurodegenerative pathology, and a better understanding of the role of various forms of amyloid plaques [the abnormalities in the brain that, along with neurofibrillary tangles, define Alzheimer's]," he adds. "With cell-based and organismal model systems, we're coming to a better understanding of the early toxic process. This improved understanding establishes the disease function of new targets, and stimulates our development of improved HTS assays to find drugs that attack neurodegeneration from these new angles."

Ruyi Hoa, director of protein development at R&D Systems, echoes that optimism. "For Alzheimer's, finding that the accumulation of beta amyloid is related to pathology is important," she says. "It has stimulated research on developing inhibitors to the enzymes responsible for producing beta amyloid peptide."

Treatments are also developing. "We're seeing the potential for advance of a lot of disease-modifying therapies," Black says. "New techniques, such as those which detect the presence of amyloid pathology in living patients, are helping to detect the disease at earlier stages and to differentiate it from other dementias. This will become increasingly important as we develop treatments to address the underlying disease process, rather than just treating symptoms."

Current Projects and Past History

Several other current projects hold promise for the development of diagnostic methods and treatments for neurological conditions. "On the diagnostic front, people are looking for biomarkers to indicate the disease early," explains Mary Lopez, strategic collaborations leader for molecular medicine business at PerkinElmer Life and Analytical Sciences. "Others are working on ways to delay the symptoms. There's also work on the connection between Alzheimer's and diabetes. People are also beginning to talk about Alzheimer's as type 3 diabetes."

German psychiatrist Alois Alzheimer first described the progressive and degenerative disease that attacks the brain and results in impaired thinking, erratic behavior, and memory loss in 1906. But not until 65 years later did active research on the disease began, and it was less than 20 years ago that researchers began developing solid theories on where to focus therapies. The U.S. Food and Drug Administration approved the first drug to treat the disease in 1993.

According to the World Health Organization, about 18 million people worldwide suffer from Alzheimer's disease, a figure projected almost to double by 2025. Caring for Alzheimer's patients exerts huge costs. One estimate suggests that the direct and total national cost of dealing with the disease in 2000 reached about $536 billion and $1.75 trillion, respectively. And families and friends who take care of patients experience emotional, physical, and financial stresses that are impossible to quantify.

Scientists have compelling evidence that genetic predisposition underlies the development of the disease. Rare cases, often with an early age of onset, are caused by dominant genes that run in families. Mutations in the amyloid precursor protein gene and presenilin-1 or presenilin-2 genes have been documented in some families. The presenilins are essential components of the proteolytic processing machinery that produces beta amyloid peptides through cleavage of amyloid precursor protein. The disease is definitely linked to the 1st, 14th, or 21st chromosomes. While researchers have identified a gene, ApoE4 on chromosome 19 that predisposes to the most common form of Alzheimer's, late onset disease, it also seems to involve other risks and protective genes, as well as environmental factors.

Tantalizing Results

Morrison-Bogorad sums up the current state of research on Alzheimer's disease. "We've been able to move from looking at the later stages of Alzheimer's to understanding the early pathology," she says. "We have a much better idea of mild cognitive impairment with memory decline, which leads to Alzheimer's in three to eight years after it's diagnosed in most cases. Alzheimer's centers have combined the clinical, pathological, and basic science into one place; interdisciplinary research in these places has generated great excitement. We have also seen very recent evidence that puts the growth factor gene at the center of some dementias. And we have identified such potential risk factors for Alzheimer's as heart disease, diabetes, and even sleep disturbances. We have a number of studies going on in these areas to see if very tantalizing results from animal and epidemiology studies are borne out by clinical trials."

As they set out to follow up on those advances, researchers in the field face several tough challenges. "The major one is identifying the early events that initiate disease and are involved in disease progression. This is crucial for developing therapies," Morrison-Bogorad explains. "Added to that is how you identify people very early as they develop Alzheimer's. You need knowledge of who to treat as well as what to treat with. And third, and increasingly important, is to understand how changes in the brain and body that result from aging contribute to Alzheimer's."

Difficulties abound at both the basic and clinical levels. "You have problems getting a diagnosis that's accurate," Tusé says. "By the time tests show definite signs of Alzheimer's, the disease may be entrenched. And retrieving samples from neural tissue is not something that you can do easily." Richard Eglen, head of R&D reagents at PerkinElmer Life and Analytical Sciences, highlights a problem in the lab. "The first issue is identifying some of the targets for treatment of the disease," he says. "Many of the early receptors have proved disappointing. There's also the difficulty of setting up clinical trials. Patients tend to die before the trials are over."

Microscopes, Cells, and Tissues

The complexity of the human nervous system, which consists of more than a trillion nerve cells, helps to explain such difficulties. Interconnections between these cells complicate the puzzle further. To explore those issues, researchers turn to a traditional tool: the microscope. While once relatively crude and cumbersome, these instruments have morphed over the years into laboratory tools that are easy to use and able to capture highly detailed images of cellular and subcellular components. "Technically, imaging systems have become much more user-friendly and much more reliable," Leica's Kier says.

Other types of microscopy for studying cells and cellular components include phase- contrast, dark-field, and fluorescence microscopy. "Multiphoton imaging is very important for the neurosciences in particular," Kier continues. "And live cell studies and calcium imaging, which involve detecting and understanding very fast events, require the speed of a digital camera or fast confocal scanners." Other major producers of microscopes include Carl Zeiss, Nikon, and Olympus. Like Leica those vendors also offer analytical software for data analysis.

In addition to microscopes, the study of living cells requires cell culture media and reagents. To keep cells in culture alive and well during experiments to study the response of nerve and other cells to changing environments, researchers and manufacturers have developed growth media and growth factors. Companies that offer those products include ATCC, Invitrogen, R&D Systems, and StemCell Technologies.

Several vendors also supply cells and tissue for research use. Asterand provides human tissue samples for neurological and cancer research, while Cambrex offers both cell systems and related products like cell culture media and sera.

The nervous system consists of a number of different cell types, including neurons, astrocytes, oligodendrites, and Schwann cells. Each type of cell possesses unique markers that scientists can use to identify it and separate it from other types. Antibodies to these markers are ideal for differentiating the neural cells. Researchers can also tag antibodies with different labels for multiplexed (simultaneous labeling) experiments.

Applications of Antibodies

Researchers also study antibodies to understand better the role they play in the process and progression of disease. Several scientific teams use antibodies tagged with labels such as fluorescein and other molecules that allow them to identify and locate specific proteins in or on a cell. These antibodies can also find application in histochemical applications, in which sections of the cell fixed in paraffin are stained with antibodies against a specific molecule. Teams can identify the tagged cells using microscopy, fluorescent readers, or flow cytometers. Scientists can also use antibodies generated to a specific protein to "find" that unique protein in a cellular extract. "The antibodies have to be specific, to help recognize the molecules," R&D Systems' Hao explains.

Companies such as Chemicon, Dako, R&D Systems, and Upstate provide antibodies tagged with markers to eliminate the need for conjugating the antibody with a label. "We have monoclonal and polyclonal antibodies; we have more than 6,000 antibodies, raised against 14 different species," Hao says. "Many have multiple applications."

Neurochemicals, certain biochemicals, and bioactive peptides represent more specialized tools applied to neuroscience. Early corporate entrants to the field, such as Biomol and Tocris Cookson, provided purified biochemicals and reagents specially tested for use in cell signaling research. To complement their reagents, those vendors offer kits for studying apoptosis, protein phosphorylation, and gene regulation.

Cayman Chemical Company and EMD Biosciences offer a wide range of kits and reagents for biochemical assays and neuroscience studies. These products combine all the materials needed to study a particular cellular function and eliminate some of the unknowns involved in sourcing different reagents and biochemicals from various providers. The suppliers have use-tested most of their kits to ensure that users can learn quickly how to conduct such experiments.

Many of the drugs originally developed as treatments for disease also have value for basic research in neuroscience. The reason: Those drugs have properties of great interest to laboratory researchers. Indeed, some of the drugs that fail clinical trials turn out to be valuable reagents in basic research. They can, for example, allow scientists to study the function of a particular biomolecule. Several companies acquire drugs of this type from major pharmas and offer them as research reagents. Providers of pharmacologicals include Axxora, BD Biosciences, and MP Biomedicals.

Exploring Living Cells

Most traditional assays require a purified cell extract, which can take several hours to prepare and must be done with great care to avoid altering the intracellular contents of a living cell. Investigators must ensure that they don't degrade or change molecules with the mechanical forces they might use to break open a cell. They must also avoid enzymatic degradation of proteins and nucleic acids via native DNase, RNase and protease molecules.

Several companies have responded to these concerns by creating systems that allow researchers to examine intact living cells in cell-based assays. "Development of these assay systems is very tightly coupled with our understanding of the disease mechanism and disease pathways," Invitrogen's Jackson explains. "Once you have a model, you can develop cellular assays that report on the activity of specific disease targets and pathways. You can work out what the pathways are in your model, and then disease researchers can make sure that what you discovered in the cell-based model also applies to the human disease."

In addition to Invitrogen, companies such as BD Biosciences, Cellomics, and PerkinElmer Life Sciences have designed systems that can process large numbers of living cells under relatively natural conditions to examine molecular interactions in cells. These systems expose cells to a compound of interest to see if the compound interacts with the living cells.

"We have developed primarily fluorescence-based assays for discovery," Jackson says. "We've been able to engineer assays incorporating target-specific beta lactamase reporters and membrane potential sensors to address many disease pathways." At present, Invitrogen's assays focus on target-specific assays aimed at many disease areas, including some assays with specific relevance to diseases such as Alzheimer's and Parkinson's. PerkinElmer's UltraView live cell system, meanwhile, "allows us to look at the appropriate response we're measuring," Eglen says. "It's a highly sensitive measurement, so you can see very small changes."

Testing Biomarkers

An increasingly important theme of neurological studies involves biomarkers. "The tools we are focusing on are for noninvasive testing of biomarkers, through blood and urine," says PerkinElmer's Lopez. "In diseases where diagnosis is a problem, a valid assay using samples obtained by noninvasive means is important," Predictive Diagnostics' Tusé adds. "Samples of blood, saliva, tears, and urine are much more important than others."

The two companies recently used a simple blood test to identify a series of biomarkers that appear to differentiate between individuals with Alzheimer's disease and those without cognitive impairment. PerkinElmer's BioXPRESSION Biomarker platform and Predictive Diagnostics' proprietary Biomarker Amplifier and Filler (BAMF) technology combined to analyze the blood in such a way as to identify patterns of proteins and peptides that distinguish Alzheimer's patients from those without clinical signs.

Both partners have moved on to more advanced methodology. "We have another relationship that we are pursuing more actively with Nonlinear Dynamics; we co-developed the mass spectrometry program PG 600 with them. The program allows you to compare rich data sets from mass spectrometry and look for specific markers," Lopez says. Predictive Diagnostics has replaced BAMF with what it calls profile technology. "It consists of machine learning tools combined with algorithms," Tusé explains. "We have set our sights on autoimmune diseases and Alzheimer's, for which we found promising biomarkers in blood samples." In collaborative work with Harvard Medical School and Tufts University, the company has shown that the profile technology is both robust and highly sensitive.

Drugs against Neurological Diseases

Several pharmaceutical firms, including GlaxoSmithKline, Lexicon Genetics, and Pfizer, have begun to develop drugs to combat Alzheimer's and other neurological diseases. Wyeth has a multiplatform approach that includes small molecule? based, peptide-based, and antibody-based platforms. Drugs under development include gamma-secretase inhibitors that reverse memory deficits in some model animals, PAI-1 inhibitors that reduce plasma and brain levels of beta-amyloid in mouse models, and active immunization programs that alter the progression of disease.

Why such a variety of approaches? "This is a hard target," Black says. "There's a lot of unexplained biology that you have to dig through in preclinical drug development. As we gain more understanding of the genetic causes of the disease and its pathology, we may be better able to tailor our therapies to individual patients with Alzheimer's disease."

Wyeth is also partnering with biotech companies Curis, Elan, and Sienabiotech to speed up the drug discovery and development process. "We acknowledge that our own basic discovery compounds are not the only sources of really exciting compounds," Black says. "We have our homegrown compounds, which we're very proud of, and we've made a partnership with Elan because it's an exciting potential treatment."

What will come next in basic and clinical research on Alzheimer's and other neurological diseases? "Testing of the amyloid hypothesis will come soon," the National Institute on Aging's Morrison-Bogorad says. "We will develop markers for evaluating therapies and drug trials. We'll develop some of the promising translational approaches we're funding. We'll look at amyloid in living patients using positron emission tomography. And we need to work on delivering drugs to the brain through the blood-brain barrier."

Peter Gwynne (pgwynne767@aol.com) is a freelance science writer based on Cape Cod, Massachusetts, U.S.A. Gary Heebner (gheebner@cell-associates.com) is a marketing consultant with Cell Associates in St. Louis, Missouri, U.S.A.

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