Initial Report on Glutamate Transporter Research

November 2, 2004

First steps towards an ALS gene therapy approach with a glutamate transporter molecule were reported on Saturday, October 23 by Christine Haeneggeli, M.D., recipient of The Milton Safenowitz Post-Doctoral Fellowship for ALS Research presented by The ALS Association.

Haenggeli and colleagues are trying to see if viral vector delivery of the glutamate transporter will delay disease in mutant SOD1 mice. Haenggeli, who gave the poster presentation at the annual meeting of the Society for Neuroscience in San Diego , is in the department of neurology and neuroscience at Johns Hopkins University in Baltimore, M.D., working in the lab of Jeffrey Rothstein, M.D., Ph.D.

The glutamate transporter, GLT1, sits on astrocytes that surround neurons, and it is the main means by which the brain keeps the excitatory transmitter glutamate at safe levels. GLT1 is the mouse version of the human glutamate transporter, EAAT2. Earlier studies by Rothstein and others showed elevated glutamate in ALS specifically occurs in areas affected by the disease process, such as the lumbar region of the spinal cord. Loss of GLT1 or EAAT2 in regions of the nervous system affected by the disease also occurs in a rodent model of ALS and human ALS, earlier studies show.

Haenggeli and her colleagues tested an adenoviral vector. Prior work by this group showed that this strategy works with insulin growth factor (IGF) to treat SOD1 mutant mice that mimic the human disorder (see related story from the meeting).

The researchers are carefully checking every step in the strategy for altering glutamate levels. They showed, in cell culture, that glutamate uptake increases a day after delivery of the transporter gene by the vector, carrying either EAAT2 or EAAT4. Two weeks after injection of hind limb muscle, a test gene borne by the vector is turning out its protein product, in some neurons innervating that leg.

The vector is going where it should and can deliver a test gene. Haenggeli noted that the team has to be sure the construct works before injecting SOD1 mice. Otherwise, she said, you won’t know why it doesn’t work, if it doesn’t.

A further challenge to this approach is that too much of the transporter might be harmful, another reason to proceed step by step.

The researchers are now working on the delivery of the EAAT2 gene by injection of the vector into muscle of SOD1 mice, to see if disease progression can be altered.

 

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More Study of Possible Link Between ALS and VEGF Gene Needed

October 27, 2004

Gene analysis of people in North America and in Ireland does not support an earlier report that a version of vascular endothelial growth factor (VEGF) is linked to higher frequency of ALS, according to findings presented at the meeting of the Society for Neuroscience on October 26. Carsten Russ, Ph.D., of Massachusetts General Hospital, said that not seeing the at risk haplotype does not negate the clear biological effect of VEGF in animal models of the disease.

Studies have shown that VEGF, a nutritive factor, can rescue mouse neurons from the effects of the SOD1 mutation, a cause of inherited forms of human ALS.

VEGF delivery by gene therapy in ALS mice shows a promising extension of lifespan, other scientists reported at the Neuroscience meeting. The negative finding that variations in the VEGF gene are not associated with ALS in the North American or Irish populations does not lessen the importance of the VEGF approach to ALS treatment, commented ALSA Science Director Lucie Bruijn, Ph.D. (see VGEF and Delayed Onset.) Further study is required to see if other populations might show the link between VEGF gene variants and ALS, Bruijn said.

The earlier study (see Gene Abonormalities), published in the July 2003 issue of Nature Genetics, was carried out in Europe, a much more homogeneous population than that of North America . The earlier study found that those people with one of three variations in the VEGF gene had a 1.8 times greater risk of developing ALS, compared to the risk seen in the overall population. These variations also were associated with a lower level of plasma VEGF.

Russ said that the Irish population was added to the current study to check findings in a homogeneous population, but still produced no clear association. His team intends to double check again in a Scottish sample. Scotland is a place of relatively little genetic variation in its population.

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ALS Animal Models Provide Excellent Resource for Scientists in Search of a Cure

October 24, 2004

Dr. Don Cleveland, professor at the Ludwig Institute of the University of California, San Diego, gave a Presidential Symposium at the meeting of the Society for Neuroscience, on October 24, providing a general overview of ALS and the status of several crucial lines of investigation towards new treatments. Cleveland emphasized that, despite lack of detail on the exact damage induced by mutant SOD1, scientists’ ability to pinpoint the mutation has provided an excellent set of animal models for uncovering a route to a cure.

Clearly the change in the SOD1 gene produces a toxic disruption of many aspects of cell function, according to Cleveland. These include impaired mitochondria, the cell’s power plant, and hampered ability to neutralize excitatory transmission by glutamate. Also, SOD1 mutation disrupts the inner scaffolding of the long motor neurons, which in people extend up to a meter down the arms and legs to control movement. Demands of these neurons, the largest cells in the body, may render them particularly vulnerable to any interruption in nutrient supply or to damage from toxic metabolic byproducts.

Cleveland  then presented recent experiments which prove that the surrounding neighborhood of a nerve cell is crucial to whether that cell lives or dies with ALS. His team and others have clearly demonstrated that neighboring glial cells can rescue motor neurons bearing an SOD1 mutation, if those nearby cells do not contain the mutation. New information obtained in Cleveland  ’s lab now shows that removal of mutant SOD1 from microglia substantially extends lifespan in an animal model.

For years, Cleveland has served on ALSA’s scientific review committee, recently as chair, and through this service has funded key projects that are closing in on therapy for ALS. Further studies presented at the meeting highlight progress:

• Exercise combined with gene therapy ameliorates mouse ALS
• Various strategies for gene therapy of ALS continue to show promise
• Silencing RNA messages from the gene can shut off the SOD1 mutation

Click on the above separate stories for more details.

 

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New Anti-Inflammatory Strategy Increases Survival of ALS Mice

October 21, 2004

New findings on possible inflammatory pathways to ALS provide a fitting introduction to ALSA’s coming coverage of the 34^th  Annual Meeting of The Society for Neuroscience. Following are recently published results of Kenneth Hensley, Ph.D. and colleagues at the University of Oklahoma, on LOX5 inhibitors and survival of SOD1 mice. Dr. Hensley will be updating his work at the meeting of the Society for Neuroscience in San Diego. Beginning Oct. 23, this gathering of about 30,000 researchers of the nervous system will include presentations from many ALSA funded investigators and advisory board members. ALSA will be posting on its web site daily highlights, beginning Monday, Oct 25.

[QUICK SUMMARY: A compound that blocks a key inflammatory enzyme prolongs survival in SOD1 mutant mice, with an effect similar to that of Rilutek, acting at the time of onset of the disorder.]

In the October issue of the journal of Neurochemistry, ALSA funded researchers at the Oklahoma Medical Research Foundation demonstrate increased survival in mice bearing a mutation that produces a disorder resembling human ALS, when they give the mice a compound, nordihydroguaiaretic acid (NDGA), that blocks part of the inflammatory cascade.

NDGA blocks 5-lipoxygenase (5LOX), a player in the pathway to inflammatory responses. A more popularly recognized enzyme, cyclooxygenase-2 (COX2), also mediates inflammation. COX2 is blocked by the class of drugs that includes Celebrex. LOX inhibitors are already aimed at asthma, with one drug, zileuton, on the market so far.

LOX inhibitors represent a new avenue towards developing effective ALS therapeutics. ALSA has just funded study investigator Kenneth Hensleyfor further exploration of this new anti-inflammatory strategy Neuroinflammatory aberrations of arachidonate pathway in ALS - The ALS Association.  ALSA also funds research into the role of related inflammatory mediators such as TNF alpha, and COX agents Cyclooxygenase. “TNF alpha and 5LOX are accessible targets for pharmacologic intervention” in ALS, Dr. Hensley and colleagues write in the report, noting that agents that act at these targets already exist.

The scientists found NDGA by testing 350 compounds known to modify inflammation in an assay of lab-grown glial cells that model inflammatory reactions. ALS mice exhibit increased levels of the 5LOX protein in their spinal cords, the scientist also showed. Levels of 5LOX build as symptoms develop.

Treatment with NDGA began at 90 days. SOD1 mice usually start showing motor defects at 90 to 100 days of age, and generally live 130 to 140 days—as the researchers confirmed. Late treatment that is effective in mouse models is particularly relevant to the human disease, which is usually not diagnosed until damage is done to the nervous system.

When the researchers put NDGA into mouse food, the treatment gave a median of 13 days increased survival—adding about a third longer life for the period after deficits appear. This prolonged survival time is similar to the effect of Rilutek (riluzole) in SOD1 mutant mice. Rilutek is the only FDA approved treatment for ALS.

ALSA is working on an initiative to prioritize compounds for clinical testing. This decision process will select such compounds as NDGA that have the most potential impact on ALS. Anti-inflammatory drugs are already in clinical trials for ALS, including minocycline and Celebrex.

 

Questions and Answers

Q:  Should I take anti-inflammatory drugs?

A: Some cannot block LOX and did not work in the animal testing. Off label use of drugs is not recommended. These agents have yet to prove effective in human ALS. NDGA efficacy in the model mouse is no better than that of rilutek. It may not be worth entering this drug into clinical testing against ALS.  Further investigation of other compounds that block LOX or COX may reveal more effective agents. NGDA is not currently formulated for human use.

Q:  Can I take zileuton?

A:   Off label use of drugs is not recommended, and this agent has not been tested clinically against ALS. Findings with zileuton will be discussed further at the meeting of the Society for Neuroscience, October 23 through 27. Further updates will follow.

 

PubMed Citation

The arachidonic acid 5-lipoxygenase inhibitor nordihydroguaiaretic acid inhibits tumor necrosis factor alpha activation of microglia and extends survival of G93A-SOD1 transgenic mice.

West M, Mhatre M, Ceballos A, Floyd RA, Grammas P, Gabbita SP, Hamdheydari L, Mai T, Mou S, Pye QN, Stewart C, West S, Williamson KS, Zemlan F, Hensley K.