Research Overview

Development and Neurobiology Group

Our focus is on understanding the mechanisms controlling the development, survival and function of cells of the nervous system, with the aim of applying this knowledge to develop new therapeutics to treat the injured or diseased nervous system. This year, we have been successful in achieving one of our long term goals: the identification and purification of the neural stem cell that resides in the adult nervous system. It is nearly ten years since we and a Canadian group simultaneously reported the presence of cells within the mature nervous system of mice, which had the potential to give rise to new neurons. This discovery changed the way we thought about the nervous system of adult animals and initiated the idea that the brain may be capable of self-repair following disease or injury. One of the main stumbling blocks to achieving this aim, however, has been the inability to identify the neural stem cell and to directly assess its properties. Thus, our successful purification of this cell from the adult brain has allowed us to begin solving this key problem. Further advances have been made in our understanding of the mechanisms that regulate neural cell death during development and in diseases such as stroke and multiple sclerosis.

Background

The group seeks to identify the key mechanisms regulating the development of the nervous system, with the aim of applying these discoveries to developing new technologies to prevent degenerative diseases and to promote repair and regeneration of the nervous system.

This approach is based on observations that many of the factors required for normal development of the nervous system can be used to stimulate regeneration within the adult nervous system.

The group uses both molecular and cellular approaches and has discovered several mechanisms key to the early development of stem cells within the nervous system and neuronal differentiation. In addition, the group has identified genes which regulate neural cell death during development and disease.

The relevance of these discoveries for diseases such as Alzheimer's, Stroke, Multiple Sclerosis and Neural Tube defects, as well as to neural repair and regeneration, is under intense scrutiny and the group is actively participating in clinical trials to ensure the rapid transfer of putative therapeutic agents into the clinic.

Birth, life and death of neurons

The human brain consists of an estimated 10 trillion individual neurons or nerve cells, each intricately interconnected with thousands of its neighbours via tendril-like projections called dendrites. The basic circuitry of the central nervous system - the brain and the spinal cord - is already in place when we are born.

For most of this century, scientists believed that the number of neurons in the central nervous system is essentially fixed at birth, and that neurons are not renewed or replaced in an individual's lifetime. Folk wisdom tells us our brains begin to die from the moment we are born - we begin to lose neurons in our late teens, and the rate of loss accelerates as we age.

It is now clear that both scientific and folk wisdom were wrong.

In the early 1990s, a team led by Dr Bartlett at the Hall Institute made a seminal discovery: the mammalian brain is not static, but possesses a self-renewing population of neuronal stem cells capable of dividing and forming new neurons. The most exciting implication of their discovery is that it might one day be possible to repair brains damaged by stroke, or by degenerative diseases like Alzheimer's disease, motor neuron disease or Parkinson's disease.

In addition, this group of researchers has thrown light on the function within the developing nervous system of LIF, a polyfunctional cytokine discovered in the Cancer and Haematology Division of the Hall Institute, and uncovered the basic mechanism that appears to underpin the death of neurons.

LIF and the nervous system

When the central nervous system (CNS) develops in the human embryo, neuronal stem cells create about twice as many neurons as the newborn brain requires. Once the brain's essential circuitry is in place, these surplus neurons undergo programmed cell death by apoptosis. Dr Bartlett and his colleagues have shown that LIF, in its capacity as a neuronal survival factor, may determine which neurons will be preserved, and which will die.

Working with Dr Carol Ware of the US biotechnology company Immunex, the Hall Institute researchers have been investigating the activity of LIF and its receptor using isolated neurons in culture and "knockout" mice engineered to lack key genes.

Transgenic mice lacking LIF receptors undergo apparently normal embryonic development, but die at birth. Brain dissections revealed an almost complete absence of astrocyte cells. Astrocytes form the matrix that physically supports and nourishes the brain's vast network of neurons; they also clear away used neurotransmitters.

Astrocytes begin to proliferate late in embryogenesis, after neuronal development is complete - at birth, the brain usually has similar numbers of both. The virtual absence of glial cells in the knockout mouse indicated that LIF plays a key role in the formation of astrocytes.

Dr Bartlett's team has recently confirmed the result by isolating neuronal stem cells from mouse embryos lacking the LIF receptor and exposing them in vitro to cytokines that would normally cause them to develop into astrocytes. The stem cells refused to form astrocytes, dramatically confirming that LIF is essential for the formation and survival of astrocytes.

The brain of the LIF receptor knockout mouse also shows another intriguing defect: the number of neurons in the motor area, which coordinates movement, is reduced by about 40 per cent. This finding hints that some factor that suppresses LIF may be involved in motor neuron disease.

"LIF may also be involved in the formation and survival of other specialised populations of neurons," Dr Bartlett said. "We need to perform detailed counts of neurons in other areas of the brain to see if we can pick up other defects in knockout mice."

Neural stem cells

Throughout life, our tissues are constantly renewed from small populations of specialised stem cells. Each time a stem cell divides, it gives rise to another stem cell, as well as to a daughter cell to replace a specialised cell that has come to the end of its useful lifetime.

Neuroscientists doubted the existence of such cells in the brain since the prevailing view was that the brain, once formed, could not create new nerve cells.

In 1992, however, Bartlett's group succeeded in isolating from the brains of embryonic mice cells that gave rise to neurons and astrocytes in vitro - and which also had the capacity to self-renew. Neuronal stem cells did exist!

In the same year, the Hall Institute team and a Canadian research group shared a potentially revolutionary discovery: the adult brain also contained cells that gave rise to new neurons.

"At first people were sceptical" says Dr Bartlett. "But in the past five years, neuronal stem cells have become the most studied cells in neurobiology."

In vitro experiments showed that just one of these cells could give rise to at least 500 new neurons - they were effectively immortal, the acid test for a stem cell.

"We now know the adult brain contains quite a large number of these cells, and at least one of their functions has been identified. They renew neurons in a region of the brain called the olfactory bulb, which serves the olfactory cells in the lining of the nose.

"We know these cells are being turned over very rapidly and being replaced by new neurons migrating in from the sub-ventricle area. But it only happens in this one area of the brain - the question is whether we can drive these cells to replace neurons in other areas of the brain or spinal cord that have been damaged by disease or trauma.

"So in the past four years we have been concentrating on the factors that regulate neurogenesis. The most important players appear to be the fibroblast growth factors."

The neuronal growth triumvirate

The Hall Institute team has shown that two closely related fibroblast growth factors - FGF-1 and FGF-2 - secreted within the brain, coordinate the production of new neurons from neuronal stem cells. Other agents, called heparin sulfate proteoglycans (HSPG), modulate their activity.

FGF-2 appears to be the factor that causes neuronal stem cells to divide and proliferate, while FGF-1 is the differentiation signal that transforms cloned stem cells into neurons.

HSPGs prime receptors on the target cells to respond to growth factors - unless the growth factor has been activated by an appropriate HSPG, it cannot bind to, and activate, its cognate receptor.

The picture emerging from studies of brain development in organisms as diverse as the African clawed toad, fruit flies and rodents, is that neurogenesis occurs as a consequence of de-repression- that is, neuronal stem cells are not activated from dormancy, but are actively repressed from proliferating and differentiating, until the repression mechanism is switched off.

In Drosophila, for example, a factor called delta, bound to a receptor called notch, prevents neuronal stem cells from differentiating into neurons. Mature neurons secrete delta, which inhibits differentiation of stem cells in their vicinity - a phenomenon called lateral inhibition.

If tissue is removed from the brain and grown in vitro, cells will continue to secrete notch, so no new neurons will grow from the sample - lateral inhibition may explain why researchers could not detect neuronal stem cells in vitro before 1992.

The quest ahead is to delineate whether FGF and HSPG stimulates neurogenesis by directly modulating the action of notch.

Saving neurons from suicide

In 1993, before the LIF-receptor knockout mouse had been developed at the Institute, Dr Graham Barrett and Dr Bartlett began experimenting with another technique for switching off gene products, using anti-sense oligonucleotides directed at the low-affinity NGF receptor (p75-NGFR).

The researchers expected the neurons treated with the antisense to die more rapidly than normal neurons because they were lacking the receptor. However, to their great surprise, the exact opposite happened, they survived.

"We repeated the experiment several hundred times before we believed the result," Dr Bartlett said. "Eventually, we realised the p75NGFR must be involved in transmitting a signal for programmed cell death - apoptosis. We were out on a limb, because everybody believed the receptor was involved in cell survival, not cell death.

"But around the time we published our result in 1994, another research team showed that over-expression of the p75NGFR can lead to cell death. Subsequently, it was shown that the p75NGFR belongs to the same family as FAS and tumour necrosis factor receptor (TNFR). All these molecules have a 'death' domain.

"The mechanisms by which FAS and TNFR work are now well understood - they bind to their receptors and activate downstream signals that switch on genes for aspartate-cleaving proteases. The proteases destroy a wide range of proteins in the cell, causing it to collapse."

The Institute has received R&D funding to develop antisense technology for clinical use - the advantage of the technique is that it has intrinsically low toxicity, and is inexpensive compared with drugs.

The team has already shown that anti-sense therapy works in living animals - when synthesis of the p75NGFR is blocked, axotomised neurons survive.

Understanding the role of p75NGFR also has ramifications for understanding brain degenerative diseases like Alzheimer's, Parkinson's and motor neuron disease. It is significant that while p75NGFR is expressed widely in the embryonic brain, it is not expressed in the adult brain, except in individuals with brain degenerative diseases. The highest levels are found in cholinergic neurons in the basal forebrain in Alzheimer's patients, which results in neuron death and memory impairment.

Neuronal Miswiring - Williams Syndrome and LIMK1

For more information about Williams Syndrome, see the US Williams Syndrome Association site. A child with Williams Syndrome (picture courtesy the Williams Syndrome Association site).

Individuals with a rare, inherited neurological disorder called Williams Syndrome suffer from mental retardation and an inability to visualise objects mentally - when asked to describe an everyday object such as a bus in their "mind's eye", for example, they may see it as a disconnected set of features, like scattered fragments of a jigsaw puzzle.

Williams Syndrome, which appears to be caused by mis-wiring of the brain during embryonic development, has been genetically linked to the loss of a gene called LIMK1. Consistent with its role in a neurological disorder, the LIMK1 protein is expressed chiefly in nerve cells, but is also present in the nuclei and cytoplasm of most other cells. Dr Ora Bernard has found that the loss of both copies of LIMK1 from the genome of mice results in the death of the mutant embryos. In Williams Syndrome, only one locus of LIMK1 has been lost - usually, two copies of every gene are present in the genome, one from each parent.

Dr Bernard and her collaborators have recently discovered that LIMK1 is intimately involved in the regulation of the actin cytoskeleton. This cellular organelle is one part of the apparatus that helps cells assume shapes appropriate for their environment; the other is the tubulin cytoskeleton. The two cytoskeletal systems are also required for the locomotion of cells, their division and for making cell to cell connections, roles of great importance in the way cells respond to their environment.

The importance of actin in cells

Actin filaments are the internal guy-wires that stabilise the shape of cells. Actin "ropes" form during mitosis, attaching themselves to the centromeres of newly replicated chromosomes. The filaments then contract, towing the chromosomes to opposite ends of the cell, so that each daughter cell ends up with an identical complement of chromosomes when the cell divides. In muscle fibres, actin literally works hand-over-hand with another protein, myosin, to effect muscular contraction. The collective action of billions of myosin molecules ratcheting over actin chains, alternately gripping and releasing, results in contraction. The mobility of macrophages, large amoeba-like cells that prowl the body scavenging for microbes and foreign organic material, depends upon actin. Structural changes in the actin cytoskeleton also enable macrophages to flow around, engulf and internalise their prey.

Actin guides the wiring-up of the nervous system

In the embryo, actively growing and dividing neurons extend thousands of thread-like projections, called neurites, that interconnect in discrete layers in the developing brain. These structures depend on a dynamic actin cytoskeleton. Actin is also an essential component in the growth cones at the extremities of embryonic neurons. As the nerve cells extend towards new targets to establish the contacts that will create neural circuits in the embryonic brain, the actin cytoskeleton provides a dynamically adjustable structure which allows growth cones to turn towards these targets. Neurons lacking a protein vital both to their structure and movement may not make the precise connections required for normal brain function.

"Perhaps in Williams Syndrome, something goes wrong and the growth cones fail to make proper contact," Dr Bernard suggests.

LIMK1 is highly expressed in the hippocampus, a small structure at the base of the brain that is required for memory formation, both short- and long-term. Lowered LIMK1 activity could disrupt the formation of memories, including visual memories. This could explain why individuals with Williams Syndrome have difficulty recalling and reintegrating the various visual aspects of a familiar object into a coherent whole.

The mechanism of LIMK1 effects

Dr Bernard and her co-workers have established that too much LIMK1 in a cell leads to the thickening of actin fibres of the cytoskeleton. They have collaborated with researchers at Switzerland's Friedrich Miescher Institute to try to understand how LIMK1 causes this abnormality and what the consequences are for the cell.

When laboratory cell lines were transfected with high levels of the LIMK1 gene, the cells developed a grossly abnormal cytoskeletal structure and died. A special stain revealed that the actin filaments which normally stabilise the structure of a cell were disrupted. Instead of assembling into long, thin chains -the filaments of the cytoskeleton- the actin molecules clumped together.

LIMK1 and cancer

Over-expression of LIMK1 results in disruption of the actin filaments that maintain the cell's internal structure. The cell loses shape and eventually dies. This finding could help in the development new cancer therapies. Taxol, a compound which prevents depolymerization of the tubulin cytoskeleton, is already used for chemotherapy. Analogous actin depolymerization-blockers are too toxic for such use, so regulating LIMK1 could be an alternative to interfering directly with the cytoskeleton.

Multiple Sclerosis - the odd one out?

Multiple sclerosis (MS) is a chronic, often disabling neurological disease of the central nervous system (CNS) - the brain and spinal cord. It involves the degeneration of the fatty myelin sheath that surrounds neurons in the CNS.

The myelin sheath, produced by specialised cells called oligodendrocytes, normally facilitates transmission of nerve impulses in the CNS by electrically insulating nerves from the surrounding environment. In MS, patches of scar tissue form and may permanently impair nerve transmission.

Multiple sclerosis is currently incurable, and its cause is unknown. It has traditionally been regarded as an autoimmune disorder, like insulin dependent diabetes and rheumatoid arthritis. But that view is now under question, according to Dr Trevor Kilpatrick.

Dr Kilpatrick says that while MS appears to have an auto-immune component, it may be a secondary phenomenon - a sequel to a more fundamental cause.

A pointer to this possibility, he says, is that immunosuppressive drugs like cyclosporin, cyclophosphamide and azothioprine, which can alleviate rheumatoid arthritis and slow the onset of insulin-dependent diabetes mellitus, are ineffective therapies for multiple sclerosis.

Researchers are exploring three possible causes of MS:

• A virus infection
• An autoimmune disorder
• An intrinsic (genetic) abnormality in the oligodendrocytes that produce myelin within the central nervous system.

"Whether it is a viral, immune-system or genetic phenomenon, we know it involves the loss of oligodendrocytes and our approach is to determine what controls the survival and death of oligodendrocytes, the ultimate aim being to develop therapies to inhibit the death of these cells in MS"

A suicide signal?

"Cell suicide or apoptosis may be involved in two ways in MS. First, episodes of remission and relapse in MS are thought to correlate with the death or proliferation of pathogenic T-cells. And apoptosis of the oligodendrocytes themselves may account for the symptoms of MS."

The Hall Institute team has focused on the role of the p75 NGF receptor family of proteins in the life and death of oligodendrocytes. The p75 NGFR family also includes the tumor necrosis factor receptor (TNFR) and Fas.

"Our interest was stimulated by a recent report that oligodendrocytes die when stimulated by nerve growth factor (NGF)" Dr Kilpatrick said. "We postulated that, in the presence of NGF, the p75NGFR receptor transmits a signal that causes oligodendrocytes to die. We have since shown that NGF enhances oligodendrocyte survival since, unlike neurons, the p75 NGFR is normally expressed at very low levels. Although we now know that products of imflammation can increase this level significantly."

Could NGF and related neurotrophic molecules promote survival of damaged oligodendrocytes and benefit patients with multiple sclerosis?

"We must be very careful," says Dr Kilpatrick. "If damaged oligodendrocytes in MS patients are induced to express high levels of p75NGFR, as reported, NGF therapy could kill the cells, which would be counter-productive. The therapy required would be to reduce p75NGFR levels either by antisense oligonucleotide treatment or by blocking the production of inflammatory proteins which regulate p75NGFR levels."

Gene therapy for MS?

Gene therapy may eventually be used to help MS patients and, according to Dr Kilpatrick, the challenge is to develop vectors that target specifically to the areas of myelin loss in the brain.

He is experimenting with a mouse model, in which lymphocytes specifically target oligodendrocytes in the brain, inducing an inflammatory response similar to MS and destroying myelin.

"Our idea is to to genetically modify these lymphocytes so they have the capacity to express proteins that will enhance the survival of oligodendrocytes by blocking the action of harmful inflammatory products. Our ultimate aim would be to develop a similar therapy for human MS patients," Dr Kilpatrick said. "We would isolate lymphocytes that react against myelin, genetically modify them, and when the patient relapses, inject them into the brain to promote oligodendrocyte survival."

Epidemiology and genes in MS

Dr Kilpatrick is also collaborating with Dr Simon Foote's group in an attempt to identify genes that may render people susceptible to MS.

A puzzling aspect of MS is that its incidence appears to correlate with latitude - in Australia, the disease is more prevalent in cooler, higher-latitude regions such as Tasmania, and less common in the tropical north. Similar gradients have been reported in Europe and North America.

Dr. Kilpatrick says "It is not clear whether this latitudinal gradient is due to an environmental agent or whether it is explained by a founder effect - the tendency of large populations to retain the original genetic imprint of a small founding population. However, there is mounting evidence for a genetic component to multiple sclerosis."

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Updated 02:55 PM (EST) on Monday, November 4, 2002.

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