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.

References:
Beers DR, Henkel JS, Zhao W, Wang J, Appel SH. CD4+ T cells support glial neuroprotection, slow disease progression, and modify glial morphology in an animal model of inherited ALS. PNAS 2008 Sep 22. [Epub ahead of print] Abstract

Suzuki M, McHugh J, Tork C, Shelley B, Hayes A, Bellantuono I, Aebischer P, Svendsen CN. Direct muscle delivery of GDNF with human mesenchymal stem cells improves motor neuron survival and function in a fat model of familial ALS. Mol Ther. 2008 Sep 16. [Epub ahead of print] Abstract

Rutherford NJ, Zhang YJ, Baker M, Gass JM, Finch NA, Xu Y-F, Stewart H, Kelley BJ, Kuntz K, Crook RJP, Sreedharan J, Vance C, Sorenson E, Lippa C, Bigio EH, Geschwind DH, Knopman DS, Mitsumoto H, Petersen RC, Cashman RR, Hutton M, Shaw CE, Boylan KB, Boeve B, Graff-Radford NR, Wszolek ZK, Caselli RJ, Dickson DW, Mackenzie IR, Petrucelli L, Rademakers R. Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis. PLoS Genet. 2008 Sep 19;4(9):e1000193. Abstract