27 Sep 2008

Recent evidence points toward glia, rather than the motorneurons themselves, as cells gone terribly wrong in promoting the pathology that drives amyotrophic lateral sclerosis (ALS), also known as motorneuron disease. Alternatively, glia may redeem themselves in certain situations and act as protective saviors, possibly even aiding in ALS treatment (see ARF related news story). Two new studies address the role that glia, immune cells, and interactions between the two, may play in ALS etiology. The first examines the ability of CD4+ T cells to modulate possible neuroprotective glial responses against mutated superoxide dismutase 1 (mSOD1). A second focuses on the ability of mesenchymal cells producing glial-derived neurotrophic factor (GDNF) to reduce disease progression when injected into the muscle of a rat model of ALS. Addressing what starts ALS pathology in the first place, a recent third report identifies new mutations in TAR DNA-binding protein 43 (TDP-43) that appear to be linked to the disease.

Dominant SOD1 mutations are the most common cause of familial ALS (Rosen et al., 1993). Expressing mutated SOD1 (mSOD1) in either neurons or glia causes motorneuron degeneration (Nagai et al., 2007; Di Giorgio et al., 2007), while diminishing both astroglial and microglial mSOD1 production slows down ALS progression (Yamanaka et al., 2008; Beers et al., 2006). For these reasons mSOD1 transgenic mice are a common model used to study ALS. The function of T cells in ALS neurodegeneration is less obvious, though some studies have indicated that T cells may have a neuroprotective function, possibly by acting on microglial responses (for review, see Schwartz et al., 2006).

A study published in this week’s Proceedings of the National Academy of Science by Stanley Appel and coworkers at the Methodist Neurological Institute, Houston, Texas, examined the role that CD4+ T cells play in disease progression and in the modification of glial responses in an animal model of ALS. Joint first authors David Beers and Jenny Henkel bred transgenic mice overexpressing mSOD1 (carrying the G93A mutation) with immunodeficient mice that lack functional CD4+ T cells. They examined the resulting crosses (mSOD1/PU.1-/- mice) for ALS pathology. These mice normally die between 15-20 days of age without a bone marrow transplant (Beers et al., 2006).

The investigators first examined the consequences of transplanting the immunodeficient mice with bone marrow from either wild-type or CCR2-/- mice. The CCR2 receptor is needed to attract activated T cells to sites of injury and is crucial for a fully functional immune system. The mSOD1/PU.1-/- mice transplanted with wild-type bone marrow had longer survival times and disease duration than the mice transplanted with CCR2-/- bone marrow, suggesting that T cell recruitment helps attenuate ALS-like disease in these animals. To investigate the role of T cells further, the researchers bred the mSOD1 with recombination-activating gene 2 knockout mice (RAG2-/- mice). These mice do not have functional T or B cells. The mSOD1/RAG2-/- mice had a shorter lifespan and disease durations versus mSOD1/RAG2-/+ littermates, suggesting that T cells do help protect against motorneuron disease. Supporting this, the researchers found that when they gave mSOD1/RAG2-/- mice bone marrow transplants from wild-type mice or mSOD1 mice, they survived longer and had a longer disease progression.

Further examining whether T cells had been restored following bone marrow transplants, Beers and colleagues used immunocytochemistry toward CD3, CD4, CD8, and a pan T cell marker. They found that no T cells were observed in the lumbar spinal cords of untreated mSOD1/RAG2-/- mice. But following bone marrow transplant at 75 days of age (which is the time of disease onset), T cells began to appear, beginning with CD3+ and CD4+ T cells. CD8+ T cells only began to appear in late-stage disease. B cells were never detected. Because CD4+ T cells were present in the transplanted animals at all stages of the disease, the researchers decided to focus on this cell subtype. They examined mSOD1 animals bred with CD4+ T cell knockout mice and found that the disease duration and survival was similar to the mSOD1/RAG2-/- mice.

The investigators concluded that the CD4+ T cells must be responsible for the prolonged disease duration and survival seen in mSOD1 mice and a fully intact immune system. In an e-mail to ARF, Appel, the principal investigator, commented, “we think that it is probably a specific sub-type of CD4+ T cells that mediates neuroprotection by modulating glial activity…the protective sub-type of CD4+ T cells may actually not be present in later [disease] stages.” This suggests that the CD4+ T cells may provide some initial protection, but eventually this defense breaks down and animals succumb to the disease.

Supporting Appel’s idea of glial cell modulation, the scientists also found that in mSOD1/RAG2-/- mice, there was an attenuation of markers of microglial activation at final stages of the disease. Analysis of cultured microglia taken from these animals revealed that the microglia were not intrinsically dysfunctional, implying that factors derived from CD4+ T cells were no longer activating the microglia. In addition, there were declines in the mRNAs for the neurotrophic factors insulin-like growth factor-1 (IGF-1), glial-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF) in the lumbar spinal cords of these mice relative to mSOD1/RAG2-/+ or mSOD1 animals. Levels of these factors were restored with bone-marrow transplant. Levels of glutamate transporters, which can increase the survival of motorneurons, were also lower in the mSOD1/RAG2-/- mice, but these were similarly restored with bone marrow transplant. Interestingly, levels of TNFα, IL6 mRNA, and NADPH oxidase isoform NOX2 mRNA were relatively increased in the mSOD1/RAG2-/- mice, and these were attenuated with bone marrow transplant.

This study seems to indicate that CD4+ T cells shield motorneurons from death in a model of ALS by modifying glial reactivity and neurotrophic factor production. According to Appel, “our study suggests that certain sub-types of T cells, such as CD4+ T cells, may be neuroprotective, and could possibly form the basis for novel future therapies in amyotrophic lateral sclerosis as well as other neurodegenerative diseases.”

Another study suggests a related though slightly different therapeutic strategy, this time focusing on the delivery of GDNF to motorneurons in a rat model of ALS. GDNF has been used in human clinical trials for Parkinson disease. Unfortunately, disappointing results in Phase 2 clinical trials prevented this therapeutic from advancing in the drug development process (see ARF related news story). Despite this, proponents of the use of GDNF in humans remain, particularly since there is evidence that GDNF promotes axonal sprouting in human brain (see ARF related news story).

Rather than infuse GDNF directly into the nervous system, however, Clive Svendsen and coworkers at the University of Wisconsin, Madison, propose using ex-vivo techniques to save motorneurons in ALS by turning human mesenchymal stem cells into surrogate glia that produce GDNF. In a study published in the September 16 issue of Molecular Therapy, first author Masatoshi Suzuki and colleagues reported that infusing GDNF-producing human mesenchymal stem cells (hMSC) into muscle protected motorneurons and improved motor function in a rat model of ALS (SOD1G93A rats).

Prior studies from this group used transplanted neural stem cells to release GDNF into the spinal cord. Although this protected motorneurons, it did not improve limb function, since connections between the motorneurons and muscles were still lost. Masatoshi Suzuki told ARF, “the novelty of the current work is that this is a combined adult stem cell and gene therapy approach targeting skeletal muscles. Compared to the spinal cord approach, muscle is easy to access and stem cells could be generated from patients themselves, lowering the risk of an adverse immune response.”

The scientists first obtained the hMSC from human neonatal bone marrow, then modified the cells to express green fluorescent protein (GFP) using a retrovirus. They then infected the cells with a lentiviral construct encoding GDNF. They measured GDNF in the medium of infected cells versus non-infected cells and confirmed that detectable amounts of GDNF were being released only by the infected cells. They further determined that the transplanted cells could survive in mSOD1 rat muscle and release GDNF. Interestingly, a small focal injury to the muscle seemed to optimize this survival.

They then measured the effect of the transplants on neuromuscular junction endplates. They found that significantly more muscle endplate innervation occurred in transplanted rats compared to controls. Significant improvement in innervation was not observed if these rats were transplanted with hMSC that had not been infected with the GDNF construct.

To confirm that hMSC-GDNF could prevent the loss of motorneurons, they counted Nissl-stained cells and cholineacetyltransferase positive cells in the lumbar spinal cord. In hMSC-GDNF-transplanted rats or even hMSC-transplanted rats, there was less cell loss, but the loss was attenuated in the hMSC-GDNF-transplanted rats. The hMSC-GDNF rats also had the longest survival time. Interestingly, the transplants did not appear to affect the activation of astroglia or microglia in the host spinal cord, indicating that GDNF itself and not gliosis likely accounted for the protection of the motorneurons.

Finally, limb function appeared to be more intact in the hMSC-GDNF rats compared with the other two groups of animals, as tested using a Basso-Beatti-Bresnahan (BBB) locomotor-rating test. Further examination of improvements in motor function and studies in humans will provide a true test of this approach. According to Suzuki “…there are many steps [needed] before it could be tested in humans.” The authors also emphasize in their conclusions that multiple injections into several muscle groups could improve motorneuron protection and movement.

These new reports underscore the importance of non-neuronal cells in ALS pathology and point toward possible therapeutic approaches that can result from transforming glial and immune cells into neuroprotective allies. Understanding glial-immune interactions remains critical to ALS research; however, other factors contributing to ALS need to be further investigated. For example, the pathological protein TDP-43 has been associated with ALS and other neurodegenerative disease. Three mutations in TDP-43 associated with familial ALS were identified in a recent article published by Rutherford et al. in the September 19 issue of PLOS Genetics. Understanding the collaborative role of non-neuronal cell responses and specific genetic mutations and their resulting protein modifications can help in the broad understanding of ALS and may ultimately provide clues for designing treatments.—Alisa Woods

Alisa Woods is a freelance writer living in Brooklyn, New York.

Comments

1. • Thomas Moeller
     University of Washington
   • Posted: 29 Sep 2008

   T Cells to the Rescue
   During inflammation, two parts of the immune system, the "innate" and the "adaptive," work hand in hand to defend against invading pathogens. The brain is harboring its own innate immune cells called glia cells, and these cells are activated in many neurodegenerative diseases such as ALS or Alzheimer disease. The activation of the brain's own innate immune cells is a double-edged sword. It can lead to neuroprotection, and frequently does so in acute injuries such as trauma or stroke. In a more chronic setting, such as neurodegenerative disease, the innate immune activation leads mainly to a detrimental outcome. The recent publication of the Appel lab now showed that a specific type of peripheral adaptive immune cells, the CD4+ T cells, enter the central nervous system in the mouse model of ALS. Once there, they seem to reprogram the local innate immune response. This leads to a more protective environment for the motor neurons, the cell type dying off in this dreadful disease. What is so astonishing about this finding is that the CD4+ cells only need to enter in a small number to produce a big effect. While still in early stages of discovery, this venue of research might open new ways for neuroprotection in ALS and other neurodegenerative diseases.

   View all comments by Thomas Moeller
2. • Trygve Holmoy
     University of Oslo, Norway, and Department of Neurology, Ulleval University Hospital, Oslo, Norway
   • Posted: 29 Sep 2008

   Amyotrophic lateral sclerosis (ALS) and Alzheimer’s might share important pathogenic pathways, and discoveries in one of these diseases or its animal models could therefore be important for the understanding of the other. Although considered a typical neurodegenerative disease mainly affecting motorneurons, ALS is often accompanied by T cell infiltration in the corticospinal tracts of patients. The significance of this T cell infiltration is not known. However, T cells have been demonstrated to secrete neurotrophic factors, and infusion of T cells specific for a myelin antigen has been demonstrated to protect against neurodegeneration after crush injury to the optic nerve and spinal cord (2).

   In the current paper, the authors addressed the significance of CD4+ T cells in mice overexpressing mutant Cu2+/Zn2+ superoxide dismutase (mSODG93A), a widely used animal model for ALS. The mSODG93A mice develop a disease with many similarities to ALS, including T cell infiltration in the spinal cord. In this study, mSODG93A mice were bred with mice lacking recombination-activating gene 2 (RAG2), which is needed for developing functional T cells and B cells. The mSODG93A/RAG2-/- mice developed more rapidly evolving disease than mSODG93A mice. In contrast to mSODG93A with functional lymphocytes, no T cell infiltration occurred in the spinal cords of the mSODG93A/RAG2-/- mice. In a series of elegant experiments with bone marrow transplantation, the authors showed that infiltrating CD4+ T cells are neuroprotective and responsible for prolonged disease duration and survival. Bone marrow transplantation also restored the CD4+ T cell expression of neurotrophic factors. Concordant data was obtained with bone marrow transplantation to mSODG93A mice from mice lacking chemokine receptor 2 (CCR2), which is needed for T cell attraction. The infiltrating lymphocytes were CD4+ T helper cells; no B cells were observed and CD8+ cytotoxic T cells were only observed at very late stages.

   How do these highly convincing data translate to human disease? This question is open to speculation, and although it is tempting to believe that the T cell infiltration observed in ALS patients is part of a reparative response to neurodegeneration, there are currently no observations in humans indicating that immune dysregulation plays a primary role in the development of ALS. T cell infiltration during early phases of ALS is extremely difficult to address, and has so far not been studied (3). Nevertheless, T cell-based therapies with glatiramer acetate (GA), an immunomodulator widely used for the treatment of multiple sclerosis, has been investigated in preclinical and early clinical trials in ALS (4,5). This drug induces an anti-inflammatory phenotype and production of substantial amounts of brain-derived nerve growth factor (BDNF) in GA-reactive T cells (6). Although the results of this therapy in humans have so far been disappointing, the results provided by Beers et al. support that T cells may be therapeutic targets in ALS. Moreover, it provides new molecular insight into the expanding field of protective immunology, showing that the T cells are not always the bad guys.

   References:

   McGeer PL, McGeer EG. Inflammatory processes in amyotrophic lateral sclerosis. Muscle Nerve. 2002 Oct;26(4):459-70. PubMed.

   Moalem G, Leibowitz-Amit R, Yoles E, Mor F, Cohen IR, Schwartz M. Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy. Nat Med. 1999 Jan;5(1):49-55. PubMed.

   Holmøy T. T cells in amyotrophic lateral sclerosis. Eur J Neurol. 2008 Apr;15(4):360-6. PubMed.

   Gordon PH, Doorish C, Montes J, Mosley RL, Mosely RL, Diamond B, Macarthur RB, Weimer LH, Kaufmann P, Hays AP, Rowland LP, Gendelman HE, Przedborski S, Mitsumoto H. Randomized controlled phase II trial of glatiramer acetate in ALS. Neurology. 2006 Apr 11;66(7):1117-9. PubMed.

   Habisch HJ, Schwalenstöcker B, Danzeisen R, Neuhaus O, Hartung HP, Ludolph A. Limited effects of glatiramer acetate in the high-copy number hSOD1-G93A mouse model of ALS. Exp Neurol. 2007 Aug;206(2):288-95. PubMed.

   Chen M, Valenzuela RM, Dhib-Jalbut S. Glatiramer acetate-reactive T cells produce brain-derived neurotrophic factor. J Neurol Sci. 2003 Nov 15;215(1-2):37-44. PubMed.

   View all comments by Trygve Holmoy

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References

News Citations

1. Glia—Absolving Neurons of Motor Neuron Disease 23 Apr 2007
2. End of the Road for GDNF Parkinson Therapy—or Just a Detour? 6 Oct 2004
3. GDNF Powers Neuron Sprouting in Human Brain 7 Jul 2005

Paper Citations

1. Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O'Regan JP, Deng HX. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993 Mar 4;362(6415):59-62. PubMed.
2. Nagai M, Re DB, Nagata T, Chalazonitis A, Jessell TM, Wichterle H, Przedborski S. Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons. Nat Neurosci. 2007 May;10(5):615-22. PubMed.
3. Di Giorgio FP, Carrasco MA, Siao MC, Maniatis T, Eggan K. Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model. Nat Neurosci. 2007 May;10(5):608-14. PubMed.
4. Yamanaka K, Chun SJ, Boillee S, Fujimori-Tonou N, Yamashita H, Gutmann DH, Takahashi R, Misawa H, Cleveland DW. Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis. Nat Neurosci. 2008 Mar;11(3):251-3. PubMed.
5. Beers DR, Henkel JS, Xiao Q, Zhao W, Wang J, Yen AA, Siklos L, McKercher SR, Appel SH. 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.
6. Schwartz M, Butovsky O, Brück W, Hanisch UK. Microglial phenotype: is the commitment reversible?. Trends Neurosci. 2006 Feb;29(2):68-74. PubMed.

Further Reading

News

1. Glia—Absolving Neurons of Motor Neuron Disease 23 Apr 2007
2. End of the Road for GDNF Parkinson Therapy—or Just a Detour? 6 Oct 2004
3. GDNF Powers Neuron Sprouting in Human Brain 7 Jul 2005