From: Roberta Friedman, Ph.D., ALSA Research Department Information Coordinator

RNA and Gene Reading: Role in ALS a Focus at Neuroscience Meeting

The mutant protein causing some inherited forms of ALS may interact with a protein that helps read DNA, as the two proteins appear to clump together in lab grown cells. According to results published in September by ALSA funded investigator William Schlaepfer, M.D., in Human Molecular Genetics, the transcription factor called HoxB2 sticks to mutant copper-zinc superoxide dismutase (SOD1) and the two proteins are found together in abnormal protein aggregates in the brain and spinal cord of mice with the SOD1 mutation. Schlaepfer will chair a symposium at the upcoming annual meeting in Washington D.C. of the Society for Neuroscience on the role of RNA in neurodegenerative diseases including ALS.

Alsin and Axonal Function, New Clues to ALS

A possible answer to the long standing puzzle as to the role of the protein alsin in ALS is published by Christopher C. J. Miller Ph.D. and colleagues at King’s College in London, who have shown that the protein, altered in a juvenile-onset ALS, may guide the growth of nerve fibers. As reported in the Oct 14 Journal of Biological Chemistry, alsin is found together with a signal protein called Rac that helps nerve fibers extent to their targets. Both proteins were located in the growth cones of axons, the researchers determined. They also showed that alsin promoted the growth of developing nerve fibers.

New insights into alsin’s possible function and how its defects could produce ALS are also provided by ALSA funded researcher Jean-Pierre Julien, Ph.D. and colleagues. They find the alsin protein in the centrosome, the part of the cell that produces the microtubule proteins critical to helping cells manage needed materials. In motor neurons, these crucial proteins allow nerve fibers to function and maintain far flung supply lines between the cell body and the target cell. As published in the August 15 issue of Biochimica et Biophysica Acta, the Canadian team produced too much alsin in monkey cells, which accumulated products instead of handling them properly. Internal organelles such as the mitochondria and Golgi apparatus also appeared damaged in these cells. Alsin was located in centrosomes in human lab grown cells as well as in a centrosome preparation purified from human

Still another clue to alsin’s role is the discovery of the genetic defect that produces a mouse that mimics some aspects of motor neuron disease. Although the wobbler mouse was discovered nearly 50 years ago and was used to model ALS prior to the design of the SOD1 mutant rodents, only now have researchers determined the exact gene defect in this spontaneous mutation. In an advance online publication in Nature Genetics, October 23, Harald Jockusch, Ph.D., University of Bielefeld, Germany and colleagues show the change is in the Vps54 gene. The protein produced by the gene is involved in handling and secreting cellular materials and interacts with microtubules. The mutant mice show damage to motor neurons and the sperm cells. As the researchers noted, alsin mutation also affects vesicular traffic, as does a rare, ALS producing mutation in a vesicle associated protein (abbreviated as VABP) that helps move materials within cells.

ALSA continues to fund research into the role of alsin and axonal transport in ALS, in hopes of finding new targets for therapeutic intervention.

Aiding Repair of Nerves

ALSA-funded researcher Stephen Strittmatter, M.D., Ph.D., published new information on why the nervous system has trouble repairing itself in the September 30 issue of Science. He and colleagues at Yale University used the visual system of mice as a model of how repair can take place. Connections among the neurons that allow mice to see are established soon after birth. After this so called critical period, experimental manipulations can no longer change how the eyes see. This critical period of flexibility in the mouse visual system is prolonged in mice that lack the receptor for Nogo, an inhibitor that prevents nerve fibers from repairing themselves after injury. The Nogo receptor is a promising therapeutic target for helping nerves to regrow. With ALSA funding, Strittmatter is continuing his efforts to understand nerve repair by studying the Nogo receptor.

Existing drugs may be able to help repair damaged nerve fibers by sidestepping inhibitors such as those acting through Nogo. The receptor for epidermal growth factor (EGF) appears to also get signals from the Nogo receptor. Drugs that block the EGF receptor, developed for cancer, were able to promote new growth of injured nerves in laboratory tests in mice, according to findings by a team led by Zhigang He, Ph.D. The tests were in the optic nerve, so research is needed to see if the same properties hold for motor neurons in order to aid therapies aimed at ALS. Genentech, a collaborator, is now looking at the action of one of the drugs in animal models of spinal cord injury, according to an accompanying news article in the journal Science.

Cell Metabolic Factor Implicated in ALS

Researchers in Uruguay published findings this past summer that a protein called Nuclear factor erythroid 2 related factor 2 (Nrf2) can help motor neurons survive. When the factor is boosted in the supportive cells called astrocytes, motor neurons growing in lab dishes alongside those astrocytes live longer. In rats with the mutant SOD1 protein, astrocytes from the lumbar spinal cord show increased Nrf2, perhaps as part of an attempt to overcome the damage, according to the report in the Journal of Biological Chemistry by a research team led by Luis Barbeito M.D., of the Institute of Biological Investigations in Montevideo. Boosting the amount of Nrf2, as the researchers did, may be a new route to treatment.

British researchers also have evidence linking Nrf2 with ALS. Pamela Shaw, M.D. of the University of Sheffield in Britain and colleagues looked at which genes are active in motor neurons growing in the lab that express mutant SOD1. The investigators found a marked decrease in activity in a group of protective genes that are controlled by Nrf2, as well as changes in activity of other genes already suspect in the biology of ALS.

Converging lines of evidence therefore implicate Nrf2 and the protective genes that it controls in the disease process of ALS. ALSA funded investigators are actively pursuing this important new lead towards correcting the damage in ALS.

Mutant SOD1 Molecules Are Marked for Cellular Trash

Mutations linked with some inherited forms of ALS produce a molecule that is marked for destruction by the cellular trash disposal system called the proteasome. As reported by Rodney L. Levine, M.D., Ph.D. and colleagues in the September 29 online issue of the Journal of Biological Chemistry, purified, metal free versions of the enzyme appear to be more readily destroyed, even without tagging by ubiquitin, which typically marks proteins for destruction. The single molecule form of SOD1, mutated or not, is more readily handled than the normal version which exists as a pair. The researchers suspect that the unstable, mutant molecule reveals water repelling regions that provide a better target for the proteasome. ALSA funded researchers continue to probe the structure and properties of mutant SOD1 to help find a way to correct defects that produce ALS.

Mutant SOD1 Abnormalities Revealed

Peering into mutant SOD1 molecules with nuclear magnetic resonance technology, an international research team showed loss of stability in solution, as the mutated form no longer appears to be able to stay together in its usual paired state. In the October 28 issue of the Journal of Biological Chemistry, the investigators, led by Joan Selverstone Valentine, Ph.D., at the University of California , Los Angeles , carefully studied a mutant SOD1 while preserving its usual points of contact within the molecule and between the molecules, as much as possible. They concluded that the mutated molecule, retaining all of its metal ions, has unstable features that make it prone to stick to its fellows.

Chick Embryo Provides Peek at ALS Disease Process

ALSA funded investigator Ray Roos, M.D. and colleagues published on the advantages to using chick embryos to study the changes in motor neurons that may produce ALS. As published on line August 4 in Neurobiology of Disease, they can use electric current to deliver gene constructs that code for different forms of the mutant SOD1 protein directly into the spinal cord of the living chick embryo. Both the mutant protein and abnormally short versions of the normal protein aggregated in the cells of the embryo and killed those cells. The model should provide a helpful way to study the toxicity behind SOD1 mutation and provide a means to test new therapeutic candidates.

Details on How IGF-1 Aids ALS Rodent

The trophic factor called IGF-1 can extend life for rats that have the SOD1 mutation by a gene therapy approach and also by direct, continuous infusion around the spinal cord. Now Japanese researchers at Okayama University led by Koji Abe, M.D., Ph.D., publishing October 18 in the Journal of Neuroscience Research, show that the IGF-1 treatment works by affecting survival messages within the cell. Normally this trophic factor docks at its receptor on cell surfaces, and the activated receptor sets into play a cascade of signals that promote survival of the cell. Proteins in this cascade are over expressed in the motor neurons still surviving in the diseased ALS rat, and IGF-1 infusion appears to normalize these proteins as the survival signals are strengthened. These details on how IGF-1 exerts its helpful action will no doubt add useful information to planning effective therapy.

How Nerves Die in ALS Rat Model

Johns Hopkins researchers working with Nicholas Maragakis, M.D. investigated exactly how the ALS rat model progresses to death. They discovered that the nerve leading to the diaphragm has loss of fibers and loss of the amplitude in its electrical signal. The diaphragm muscle shrinks. But there is no evidence that the endings of the nerve die first, a process called “dying back.” In their online report August 3 in Neurobiology of Disease, the investigators note that the rapid progress of disease in the rat may prevent dying back from becoming apparent, as compared with the mouse model. Motor neurons are relatively spared in the rat cranial nerve nuclei; most loss of motor units occurs in the lumbosacral spinal cord.

Certain SOD1 Mutations May Mean More Aggressive ALS

Researchers who delved two hundred years back into families with inherited ALS have found that a certain mutation in the enzyme copper-zinc superoxide dismutase (SOD1) may lead to earlier death from the disease with implications for genetic counseling of affected individuals. As published in August in the International Journal of Neuroscience by Australian researcher Garth Nicholson, Ph.D., who has received ALSA funding, family histories show a powerful effect of the valine to glycine change at amino acid 148 in the SOD1 protein. With this mutation, affected family members died of ALS nearly ten years earlier than other people with different mutations in the enzyme: in their 40s rather than in their 50s. Lifespan of all three families investigated was cut on average by a decade by the inherited disease, in comparison to survival time with sporadic ALS. Age of onset was not different for each of the three mutations than with sporadic ALS, Nicholson wrote, so the inherited mutation may produce a more aggressive form of ALS.

Stem Cells Struggle to Make Repairs in ALS Mice

In mice with mutant SOD1 disease, their own stem cells are making a valiant attempt to repair damage, according to research published by scientists led by Rugao Liu, Ph.D., at the University of North Dakota in Grand Forks, in the August issue of Stem Cells. During onset and progression of the ALS like disorder, the spinal cords of the mice show stem cells that are proliferating and entering the areas where motor neurons are dying. ALSA is continuing to fund the search for effective stem cell therapies in ALS.

Ways to Make Stem Cells Avoid Creating Viable Embryos

In an attempt to make stem cells that would not require stopping a nascent human life, two teams of researchers published in the October 16 online issue of Nature on two potential ways to avoid destroying an individual being while still producing stem cells suitable for therapeutics. Rudolf Jaenisch, M.D., Ph.D. of the Whitehead Institute at the Massachusetts Institute of Technology in Cambridge and colleagues created mouse embryos with a silenced gene that prevents formation of the tissue of the placenta, so the source embryo would never be able to implant into a uterus and grow. The stem cells can be harvested from such an embryo at the blastocyst stage, as is usual practice, and the gene silencing can then be reversed in those cells to produce fully functioning stem cells.

In a second paper in that issue of Nature, researchers including a group at Advanced Cell Technology in Worcester, Mass., led by Robert Lanza, M.D., used a converse approach, taking mouse stem cells but intentionally preserving the ability of the source embryo to become an individual. The researchers took a single cell, a technique routinely used by those working with in vitro fertilization to check that an in vitro embryo is genetically normal, at a time when the embryo consists of eight cells. The researchers showed the single cell could be used to derive a line of stem cells that would form all the tissues of the body. The key was to co-culture the isolated cell (called a blastomere) with other embryonic stem cells.

In an accompanying comment on both papers, stem cell researcher Irving Weissman, M.D., Ph.D. at Stanford University in California, wrote that all paths towards the scientific and therapeutic use of stem cells ought to proceed in parallel.