ALS-TDI Scours Transcriptome, Targets CD40L

After sifting through nearly 500 biological pathways, almost a dozen kinds of tissues from 100 mice, and some weighty statistical scores, the research team at the Amyotrophic Lateral Sclerosis Therapy Development Institute (ALS-TDI) in Cambridge, Massachusetts, has identified some potential drug targets. ARF’s regular readers may not be surprised that one of their strongest hits points to the immune system. In a Nature Genetics paper posted online March 28, the scientists report that blocking the interaction between CD40 and its ligand CD40L delays disease and prolongs survival in a mouse model of ALS. This damaging immune pathway appears to ramp up in humans with ALS, as well.

The researchers, led by joint first authors John Lincecum and Fernando Vieira and senior author Steven Perrin, suggest that a therapeutic antibody to CD40L has an immuno-modulatory effect, and hope to start clinical trials soon. We do not think we can do much more with this agent in preclinical animal models, Perrin said. Bench-to-bedside is what it is about.

ALS-TDI took an unbiased approach to their hunt, searching the transcriptome of an ALS mouse model for genes that are up- or downregulated as the disease progresses. The model expresses the G93A variant of human superoxide dismutase-1 (SOD1), a mutant that causes ALS in humans. The mice start to show obvious symptoms of motor problems and paralysis around 75 days of age and have a median survival of 132 days, much shorter than the typical two- to three-year lifespan of a mouse.

The researchers harvested tissue from five mutant SOD1 (mSOD1) and five non-transgenic mice every 10 days beginning at the age of 30 days—well before symptoms are readily apparent—and continuing to 120 days of age. They dissected out brain, spinal cord, skeletal muscle, sciatic nerve, blood, and adipose tissue, purified RNA, and used gene chips to compare gene expression levels among all time points and tissue types. They then analyzed how differentially regulated genes fit into known biological pathways. The experiment cost approximately $3 million, Perrin said.

Through this screen, ALS-TDI identified five biological pathways, comprising transcripts for 95 different genes, related to immune function. Of these pathways, two cause T cell activation, two activate macrophages, and one was the co-stimulatory pathway that links the innate and adaptive immune system. Large-scale genomewide screens do not always yield results commensurate with their price tags, noted Terrence Town of the Cedars-Sinai Medical Center in Los Angeles, so this is really a success story. Town was not part of the study group.

The co-stimulatory pathway is driven by interactions between antigen-presenting cells, such as macrophages, and the T cells that mediate the body’s immune response. T cell receptors such as CD40 bind to corresponding molecules on the antigen-presenting cells, amplifying the immune response. Aberrant activation of the co-stimulatory pathway may lead to inappropriate autoimmune responses.

Recent evidence indicates that neuroinflammation and the immune system are involved in ALS, but are not altogether beneficial. Microglia, the immune cells of the central nervous system, appear to have both positive and negative effects on the disease (see Henkel et al., 2009). But the immune system’s role may also extend to peripheral macrophages (see ARF related news story on Chiu et al., 2009). And not just the innate, but also the adaptive, T cell-based immune system appears to get involved (see ARF related news story). Immuno-modulatory therapies have previously been linked to motor neuron survival in mSOD1 mice (Kiaei et al., 2006).

Perrin and colleagues chose to focus on the co-stimulatory pathway because its players steadily increased along with symptoms in ALS-related tissues (muscle, spinal cord, and sciatic nerve) of mSOD1 mice, compared to wild-type animals. Further, Perrin and colleagues analyzed the activation of immune system genes in people with ALS. They found that genes in the co-stimulatory pathway were upregulated in 35 of 63 (56 percent) blood samples from people with ALS, compared to non-ALS controls.

Between the new focus on immunity and ALS, and their own results, the ALS-TDI researchers thought the co-stimulatory pathway might be a potential drug target. Blocking the CD40-CD40L interaction has already been fingered as a possible therapeutic in Alzheimer disease, since it reduces amyloid plaques in a mouse AD model (see ARF related news story on Tan et al., 2002). Interfering with CD40L binding is also beneficial in preclinical models of other conditions, such as multiple sclerosis (Gerritse et al., 1996) and arthritis (Durie et al., 1993). However, early immunosuppressive drugs that targeted the CD40 pairing had too many side effects, Perrin said. Drug design has been challenging, Town noted: CD40 and CD40L stick like molecular Velcro, so it is no wonder that attempts at drug inhibitors have not been fruitful. But newer drugs have fewer side effects, Perrin said, making the CD40 interaction an appealing target again.

To test their hypothesis that blocking CD40L could slow motor neuron disease, Perrin and colleagues treated 44 mice with a monoclonal antibody to CD40L to prevent its binding to CD40. They started treatment at day 50, before obvious symptoms present. Compared to placebo controls, treated mice experienced a six-day delay in the start of disease-associated weight loss and an eight-day delay in the onset of paralysis. The treated animals also downregulated the co-stimulatory pathway in the spinal cord, had less inflammation in the central and peripheral nervous systems, and had more motor neuron cell bodies than did their untreated counterparts.

On average, treated mice lived for nine days longer than untreated mice. The survival difference is small, but significant for mice that are so sick, said Stanley Appel of The Methodist Neurological Institute in Houston, Texas. If you can get them up to 140 [days], or a little bit longer, you have accomplished a lot, he said. Those nine days in mice might correspond to three to five years in humans, if the therapy were even effective in people, speculated Mahmoud Kiaei of Cornell University Medical College in New York City. When the researchers limited their analysis to only females, they saw an average of 13 more days of survival. It is not clear why the female mice did better on the treatment.

How does toning down the co-stimulatory pathway fight ALS pathology The ALS-TDI researchers suggest a model hinging on resident monocytes that watch over the peripheral axons of motor neurons. When some antigen—identity unknown—upregulates this monocyte surveillance via the co-stimulatory pathway, it causes an immune response leading to neuroinflammation and motor neuron damage.

Several CD40L blockers have already passed Phase 1 safety trials for cancer and other conditions, and ALS-TDI is in talks with companies that could be a clinical trial partner. Appel, who is on the ALS-TDI board of directors, is cautiously optimistic about the success of such a trial. The science is impeccable, the logic is beautiful, he said. If there is ever going to be rational therapy based on good science, here is an example of it. However, he noted that ALS researchers have been burned before with promising therapies that do not help people. Many treatments that extend survival in the SOD1 mouse fail in the clinic (see ARF Live Discussion and Scott et al., 2008), and researchers do not want to give people with ALS what may amount to false hope. As another caveat, Michal Schwartz of the Wizmann Institute of Science in Rehovot, Israel, cautioned in an e-mail to ARF that CD40 and CD40L have a variety of functions, so silencing their interaction could have both positive and negative outcomes.

Many diseases might be amenable to CD40L treatment, Town suggested. Now that ALS and Alzheimer’s have been linked to CD40L, at least in mice, it might be worth pursuing Parkinson disease or frontotemporal dementia. I think this paper opens the door, he said.

Reference:
Lincecum JM, Vieira FG, Wang MZ, Thompson K, De Zutter GS, Kidd J, Moreno A, Sanchez R, Carrion IJ, Levine BA, Al-Nakhala BM, Sullivan SM, Gill A, Perrin S. From transcriptome analysis to therapeutic anti-CD40L treatment in the SOD1 model of amyotrophic lateral sclerosis. Nat Genet. 2010 Mar 28. Abstract


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