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.—Amber Dance


  1. This article elegantly shows the strength of transcriptome analysis for the rapid discovery of a new drug. In this study, the authors identified the therapeutic potential of modulating CD40L in ALS using an animal model.

    Through transcriptome analysis, this group identified the upregulation of CD40L-related pathway in three tissues that are all relevant to motor neuron degeneration: muscle, spinal cord, and sciatic nerve. This signaling pathway related to CD40L activation became more prominent as the disease progressed; this finding justifiably led the investigators to test its implication to therapy. The therapeutic potential was tested in mSOD1 mice, and anti-CD40L was found to be effective with respect to both disease onset and progression. The authors compared the results to those observed in inflammatory diseases and, based on Mac-1 expression and T cell activation, suggested that the therapy acts in the animal model of mSOD1 as anti-inflammatory treatment; such a conclusion should be taken with caution, and more so when it comes to clinical translation.

    CD40L was originally described on T lymphocytes; its expression has since been detected on a wide variety of cells, including platelets, mast cells, macrophages, basophils, NK cells, B lymphocytes, as well as non-hematopoietic macrophages. Primarily, in its bound form, CD40L serves as a self-controlling, co-stimulatory molecule; thus, it acts as a mechanism of prevention of unnecessary lymphocyte activation and works at multiple levels. CD40L allows full immune cell activation, prevents anergy or apoptosis, induces differentiation to effector or memory status, sustains cell proliferation, and allows cell-cell crosstalk and cooperation. Therefore, neutralizing CD40L might lead to different effects at different stages of the disease. Moreover, its mechanism of action may be critically affected by the dosing, resulting in an effect suggestive of Dr. Jekyll and Mr. Hyde. This situation is very much reminiscent of minocycline in ALS, which showed similar efficacy in animal models of mSOD1 and failed in human trials. The case of minocycline might represent a general phenomenon with respect to the use of anti-inflammatory therapies in ALS. Such therapies are beneficial in inflammatory diseases such as multiple sclerosis, and, in their relevant animal model, experimental autoimmune encephalomyelitis (EAE). As opposed to ALS, these diseases are inflammatory in their etiology, whereas ALS is characterized by local inflammation, but is not considered an inflammatory disease. Moreover, elevated CD40L in mSOD1 mice might represent beneficial attempts to cope with the disease that are not sufficiently controlled. Therefore, blockage of CD40L may have a beneficial phase/effect/outcome at certain disease stages, but not in a blanket way. Thus, targeting a co-stimulatory molecule as a therapeutic approach may interrupt essential beneficial immune responses in addition to targeting the disease process.

  2. Against the backdrop of sometimes disappointing results from genomewide association studies of the transcriptome (GWAS-T), the work by Lincecum and colleagues represents a triumph for this approach. The authors applied transcriptome analysis to the high-copy SOD1 transgenic mouse model of ALS. Importantly, they thoroughly investigated central and peripheral tissues from SOD1 mice at timepoints prior to, during, and after disease onset. Their GWAS-T results pointed to co-stimulatory immune and inflammatory molecules as being centrally associated with ALS-like pathology in this system, and they utilized a sophisticated statistical algorithm to arrive at the CD40-CD40L interaction as a candidate treatment target. They then treated SOD1 mice with a neutralizing CD40L antibody and found benefit by virtually any index of ALS-like disease: the biologic therapy improved body weight maintenance and survival, reduced inflammatory lesions, decreased motor neuron loss, and attenuated expression of immune co-stimulatory genes.

    I read this work with enthusiasm and excitement, because over a decade ago we demonstrated that pharmacologic or genetic blockade of CD40-CD40L interaction mitigated AD-like pathology in transgenic mouse models of the disease. This included reduction of: abnormal tau proteins, cerebral amyloidosis, brain inflammation including gliosis, and behavioral impairment (Tan et al., 1999; Tan et al., 2002). At that time, many in the field of AD research were unwilling to accept that the immune system played any role in the pathogenesis of AD, let alone that immune molecules could be targeted for AD treatment. It is terribly exciting that these authors have extended the concepts that we were exploring vis-à-vis CD40-CD40L in AD to another key neurodegenerative disease: ALS. I hope that the authors are able to successfully translate their findings to the clinical syndrome.


    . Microglial activation resulting from CD40-CD40L interaction after beta-amyloid stimulation. Science. 1999 Dec 17;286(5448):2352-5. PubMed.

    . Role of CD40 ligand in amyloidosis in transgenic Alzheimer's mice. Nat Neurosci. 2002 Dec;5(12):1288-93. PubMed.

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News Citations

  1. Peripheral Innate Immunity—Not So Peripheral to ALS?
  2. ALS: T Cells Step Up
  3. Orlando Conference: The Chicken and the Egg

Webinar Citations

  1. Mice on Trial? Issues in the Design of Drug Studies

Paper Citations

  1. . Microglia in ALS: the good, the bad, and the resting. J Neuroimmune Pharmacol. 2009 Dec;4(4):389-98. PubMed.
  2. . Activation of innate and humoral immunity in the peripheral nervous system of ALS transgenic mice. Proc Natl Acad Sci U S A. 2009 Dec 8;106(49):20960-5. PubMed.
  3. . Thalidomide and lenalidomide extend survival in a transgenic mouse model of amyotrophic lateral sclerosis. J Neurosci. 2006 Mar 1;26(9):2467-73. PubMed.
  4. . Role of CD40 ligand in amyloidosis in transgenic Alzheimer's mice. Nat Neurosci. 2002 Dec;5(12):1288-93. PubMed.
  5. . CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2499-504. PubMed.
  6. . Prevention of collagen-induced arthritis with an antibody to gp39, the ligand for CD40. Science. 1993 Sep 3;261(5126):1328-30. PubMed.
  7. . Design, power, and interpretation of studies in the standard murine model of ALS. Amyotroph Lateral Scler. 2008;9(1):4-15. PubMed.

Further Reading


  1. . Neuroinflammation in Alzheimer's disease and mild cognitive impairment: a field in its infancy. J Alzheimers Dis. 2010;19(1):355-61. PubMed.
  2. . Thymic involution, a co-morbidity factor in amyotrophic lateral sclerosis. J Cell Mol Med. 2010 Oct;14(10):2470-82. PubMed.
  3. . T lymphocytes potentiate endogenous neuroprotective inflammation in a mouse model of ALS. Proc Natl Acad Sci U S A. 2008 Nov 18;105(46):17913-8. Epub 2008 Nov 7 PubMed.
  4. . Inflammatory processes in amyotrophic lateral sclerosis. Muscle Nerve. 2002 Oct;26(4):459-70. PubMed.
  5. . Evidence for systemic immune system alterations in sporadic amyotrophic lateral sclerosis (sALS). J Neuroimmunol. 2005 Feb;159(1-2):215-24. PubMed.
  6. . Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer's disease. Nat Neurosci. 2010 Apr;13(4):411-3. PubMed.
  7. . T cell-microglial dialogue in Parkinson's disease and amyotrophic lateral sclerosis: are we listening?. Trends Immunol. 2010 Jan;31(1):7-17. PubMed.
  8. . CD4+ T cells support glial neuroprotection, slow disease progression, and modify glial morphology in an animal model of inherited ALS. Proc Natl Acad Sci U S A. 2008 Oct 7;105(40):15558-63. Epub 2008 Sep 22 PubMed.
  9. . Wild-type microglia extend survival in PU.1 knockout mice with familial amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A. 2006 Oct 24;103(43):16021-6. PubMed.
  10. . Microglial activation resulting from CD40-CD40L interaction after beta-amyloid stimulation. Science. 1999 Dec 17;286(5448):2352-5. PubMed.

Primary Papers

  1. . From transcriptome analysis to therapeutic anti-CD40L treatment in the SOD1 model of amyotrophic lateral sclerosis. Nat Genet. 2010 May;42(5):392-9. PubMed.