In the recent papers from the groups of Przedborski and Eggan, provocative evidence is reported that spinal motor neurons may die in SOD1 mutant mice because of soluble toxic factors released by SOD1 mutant astrocytes. This result is surprising because previous studies with chimeric SOD1 mutant mice have shown that expression of mutant SOD1 in microglia but not astrocytes is implicated in the neuron death. However, profound reactive astrocytosis occurs very early in mouse and human motor neuron diseases. This is true in the SOD1 mutant mice, where reactive astrocytosis is a dramatic feature of the disease, with prominent reactive astrocytosis occurring long before much motor neuron death occurs (Carlos Pardo, personal communication).

The new studies provide striking evidence that astrocyte-conditioned medium from SOD1 mutant astrocytes is toxic, as wild-type spinal motor neurons survive longer in culture when cultured alone or with wild-type astrocyte conditioned medium than with mutant astrocyte- conditioned medium. Thus, the lower survival of the spinal motor neurons...  Read more

In the recent papers from the groups of Przedborski and Eggan, provocative evidence is reported that spinal motor neurons may die in SOD1 mutant mice because of soluble toxic factors released by SOD1 mutant astrocytes. This result is surprising because previous studies with chimeric SOD1 mutant mice have shown that expression of mutant SOD1 in microglia but not astrocytes is implicated in the neuron death. However, profound reactive astrocytosis occurs very early in mouse and human motor neuron diseases. This is true in the SOD1 mutant mice, where reactive astrocytosis is a dramatic feature of the disease, with prominent reactive astrocytosis occurring long before much motor neuron death occurs (Carlos Pardo, personal communication).

The new studies provide striking evidence that astrocyte-conditioned medium from SOD1 mutant astrocytes is toxic, as wild-type spinal motor neurons survive longer in culture when cultured alone or with wild-type astrocyte conditioned medium than with mutant astrocyte- conditioned medium. Thus, the lower survival of the spinal motor neurons cannot be attributed to less production of neurotrophic factors by the mutant astrocytes. Together, these in-vitro and in-vivo findings directly implicate reactive astrocytes in the pathophysiology of spinal motor neuron death in the SOD1 mutant mice. One caveat is that in the in-vitro studies, the astrocytes that were studied were obtained from neonatal spinal cords long before any reactive gliosis actually occurs. However, neonatal astrocytes in culture have a similar phenotype to reactive astrocytes in vivo, and may in fact be comparable.

So how can these new observations of Przedborski and Eggan be reconciled with previous studies that found that chimeric SOD1 mutant mice with mutant SOD1 in microglia but not astrocytes is implicated in the neuron death? For one thing, it is unclear if mutant microglia were actually present in the astrocyte cultures used in these new studies. Steps were taken to minimize microglial contamination, but because astrocytes secrete high levels of microglial mitogens such as colony stimulating factor-1 (CSF1), microglia almost always heavily contaminate neonatal astrocyte cultures prepared by the commonly used methods of McCarthy and DeVellis. It would be good to repeat the study using more stringent methods of microglial elimination, such as immunopanning, and it would be important to confirm by immunostaining that microglia are in fact absent from the astrocyte-conditioned medium at the time it is harvested. It is also possible that mutant astrocytes release factors in culture that are toxic to motor neurons but that these factors are not actually secreted in vivo or are not toxic to the neurons in vivo. The only way to find out for sure, of course, will be to identify this toxic astrocyte factor. At the present time, it is difficult to think of a model that reconciles the previous in-vivo observations implicating mutant microglia with the present in-vitro observations implicating mutant astrocytes.

Assuming astrocytes make a toxic factor, what could be its nature? The possibility that it is glutamate has already been ruled out, as have been the obvious cytokine candidates. Moreover, the motor neurons undergo apoptosis. One possibility is that it is a factor that binds to, inhibits, or proteolyzes required trophic factors or culture substrates present in the culture medium that are required for long-term motor neuron survival.

Another possibility is that the toxic astrocytes alter the pH of the culture medium or lower antioxidant levels, which are both crucial parameters for good neuronal survival. Alternatively, a toxic factor could be released, such as a cytokine of some sort or an excitotoxin. Glutamate agonists have not been ruled out. For instance, homocysteine is an NMDA agonist that is exclusively made by astrocytes; other possibilities are aspartate and N-acetyl-aspartylglutamate, which all act on NMDA receptors. In addition, astrocytes have previously been shown to secrete high levels of NMDA potentiators such as L-glycine or D-serine, and it is possible that the mutant astrocytes secrete higher levels of these. Glutamate excitotoxicity can lead to apoptosis, so it would be important in future experiments to test whether the toxic astrocyte factor can be blocked by APV or other NMDA receptor blockers, as so far only kainate and AMPA receptor blockers have been tested.

A very interesting new paper by Don Cleveland’s group provides evidence, using laser capture studies of mRNA expression, that spinal motor neurons in the SOD mutant mice have elevated levels of several complement proteins. This raises the possibility that there is complement-induced toxicity. However, microglia and serum, which are both rich sources of the complete set of complement proteins required for the complement cascade to function, were not present in the motor neuron cultures; therefore, this seems an unlikely possibility. Moreover, complement-mediated toxicity would be expected to cause lysis and not necessarily apoptosis (though mild toxic insults are well documented to lead to apoptosis in neurons). Interestingly, the new Cleveland work also provides evidence for a strong upregulation of the serine biosynthetic pathway. Astrocytes have previously been shown to preferentially use the serine synthetic pathway, whereas neurons do not (a result we have recently confirmed by gene profiling of purified neural cell populations; Cahoy and Barres, unpublished observations). It is possible that SOD1 mutant neurons upregulate these pathways as they die, but a more likely possibility is that there was some contamination by reactive glial genes in these studies, a possibility that is suggested by the presence of other upregulated well-described astrocyte genes such as CD44 and aquaporin 4. This latter possibility again raises the possibility that the toxic factor being released by mutant astrocytes is D-serine or L-glycine.

Whatever the case, these new papers call attention to the important but still poorly understood roles of neuron-glial interactions in the pathophysiology of neurodegenerative disease.

View all comments by Ben Barres

These two papers by Nagai et al. (2007) and Di Giorgio et al. (2007) independently provide strong evidence that glial cells, and perhaps specifically astrocytes, bearing SOD1 mutations are responsible for degeneration and death of motor neurons in embryonic stem cell (ESC)-based co-cultures of primary neurons and glial cells. Motor neurons bearing SOD1 mutation did not degenerate in the absence of mutant glial cells.

While these elegant findings provide important insights into the interdependency between neurons and glial cells, and provide key data concerning the pathogenesis of human ALS associated with SOD1 mutation, their relevance to sporadic and other non-SOD1 related forms of human ALS is uncertain. Increasingly, it is becoming recognized that SOD1- associated ALS, and non-SOD1 forms of ALS may be driven through different pathogenetic cascade mechanisms. In SOD1 ALS, the accumulated protein within the conglomerated ubiquitinated inclusion bodies is mutated SOD1. In other, non-SOD1 forms of familial ALS, and sporadic ALS, the filamentous or skein-like ubiquitinated...  Read more

These two papers by Nagai et al. (2007) and Di Giorgio et al. (2007) independently provide strong evidence that glial cells, and perhaps specifically astrocytes, bearing SOD1 mutations are responsible for degeneration and death of motor neurons in embryonic stem cell (ESC)-based co-cultures of primary neurons and glial cells. Motor neurons bearing SOD1 mutation did not degenerate in the absence of mutant glial cells.

While these elegant findings provide important insights into the interdependency between neurons and glial cells, and provide key data concerning the pathogenesis of human ALS associated with SOD1 mutation, their relevance to sporadic and other non-SOD1 related forms of human ALS is uncertain. Increasingly, it is becoming recognized that SOD1- associated ALS, and non-SOD1 forms of ALS may be driven through different pathogenetic cascade mechanisms. In SOD1 ALS, the accumulated protein within the conglomerated ubiquitinated inclusion bodies is mutated SOD1. In other, non-SOD1 forms of familial ALS, and sporadic ALS, the filamentous or skein-like ubiquitinated inclusions contain the TAR DNA binding protein, TDP-43 (Neumann et al., 2006; Davidson et al., 2007). Pertinently, the inclusions in SOD1-associated ALS are not TDP-43 immunoreactive (Tan et al., 2007). These latter morphological and immunohistochemical data reinforce the concept that SOD1 and non-SOD1 ALS are separate disorders even though they share a common clinical phenotype. The data, moreover, imply that a role for glial cells, as described in the work of Nagai et al., (2007) and Di Giorgio et al. (2007), may not pertain in the more common forms of ALS that are not associated with SOD1 mutation.

Nonetheless, a potential role for glial cells in non-SOD1 ALS could, perhaps, be tested in ESC-based studies using the Q342X stop codon mutation in the intraflagellar transport protein 74 (IFT74) gene, which has been associated in one family with a frontotemporal dementia and motor neuron disease (FTD+MND) clinical phenotype (Momeni et al., 2006). One patient from this family with this mutation showed ubiquitinated pathological changes within cerebral cortex and brain stem and spinal cord detectable by TDP-43 immunohistochemistry (Cairns et al., 2007). These were typical of those seen in FTD+MND, and in ALS alone (Neumann et al., 2006; Davidson et al., 2007).

References:
Cairns NJ et al (2007) Amer J Pathol (in press).

Davidson Y, Kelley T, Mackenzie IR, Pickering-Brown S, Du Plessis D, Neary D, Snowden JS, Mann DM. Ubiquitinated pathological lesions in frontotemporal lobar degeneration contain the TAR DNA-binding protein, TDP-43. Acta Neuropathol (Berl). 2007 May;113(5):521-33. Epub 2007 Jan 12. Abstract

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):608-614. Epub 2007 Apr 15. Abstract

Momeni P, Schymick J, Jain S, Cookson MR, Cairns NJ, Greggio E, Greenway MJ, Berger S, Pickering-Brown S, Chio A, Fung HC, Holtzman DM, Huey ED, Wassermann EM, Adamson J, Hutton ML, Rogaeva E, St George-Hyslop P, Rothstein JD, Hardiman O, Grafman J, Singleton A, Hardy J, Traynor BJ. Analysis of IFT74 as a candidate gene for chromosome 9p-linked ALS-FTD. BMC Neurol. 2006 Dec 13;6:44. Abstract

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):615-622. Epub 2007 Apr 15. Abstract

Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, Bruce J, Schuck T, Grossman M, Clark CM, McCluskey LF, Miller BL, Masliah E, Mackenzie IR, Feldman H, Feiden W, Kretzschmar HA, Trojanowski JQ, Lee VM. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. 2006 Oct 6;314(5796):130-3. Abstract

Tan CF, Eguchi H, Tagawa A, Onodera O, Iwasaki T, Tsujino A, Nishizawa M, Kakita A, Takahashi H. TDP-43 immunoreactivity in neuronal inclusions in familial amyotrophic lateral sclerosis with or without SOD1 gene mutation. Acta Neuropathol (Berl). 2007 May;113(5):535-42. Epub 2007 Feb 27. Abstract

View all comments by David M.A. Mann

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...  Read more

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

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...  Read more

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

2. 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. Abstract

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

4. 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. Abstract

5. 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. Abstract

6. 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. Abstract

View all comments by Trygve Holmoy

In light of the ongoing efforts to downregulate SOD1 via various RNA interference approaches, the recent paper by Lobsiger and colleagues has particular significance. It poignantly reminds us that not all “mutant” SOD1 is toxic—but rather some SOD1 seems to function in its intended capacity as an antioxidant enzyme. Furthermore, while we assume that all mutant SOD1-mediated toxicity must converge on a final common pathway resulting in motor neuron degeneration and ultimate death, the roads along the way might be slightly different.

In the report put forward by Lobsiger, the (efficient) removal of SOD1 from the peripheral Schwann cells yielded a very unexpected outcome—disease was accelerated. It has now been accepted that non-cell autonomous mechanisms must be at play in motor neuron degeneration, but the same is obviously true for motor neuron survival as well. Clearly, Schwann cells (which have the most intimate association with motor neurons, numbering 1000:1!) provide essential function for the maintenance of motor axons. Indeed, earlier work (Reaume et al.,...  Read more

In light of the ongoing efforts to downregulate SOD1 via various RNA interference approaches, the recent paper by Lobsiger and colleagues has particular significance. It poignantly reminds us that not all “mutant” SOD1 is toxic—but rather some SOD1 seems to function in its intended capacity as an antioxidant enzyme. Furthermore, while we assume that all mutant SOD1-mediated toxicity must converge on a final common pathway resulting in motor neuron degeneration and ultimate death, the roads along the way might be slightly different.

In the report put forward by Lobsiger, the (efficient) removal of SOD1 from the peripheral Schwann cells yielded a very unexpected outcome—disease was accelerated. It has now been accepted that non-cell autonomous mechanisms must be at play in motor neuron degeneration, but the same is obviously true for motor neuron survival as well. Clearly, Schwann cells (which have the most intimate association with motor neurons, numbering 1000:1!) provide essential function for the maintenance of motor axons. Indeed, earlier work (Reaume et al., 1996) demonstrated that recovery from axonal injury was impaired in SOD1-/- mice. However, it was assumed that the lack of recovery was due to the lack of SOD1 action within the motor neuron. What is now evident from Lobsiger’s work is that location matters: SOD1 action within Schwann cells actively participates in axonal recovery and maintenance.

While future experiments using the mentioned floxed G85R mouse will be the direct test of this hypothesis, this is an opportunity for reflection in ALS. At present, multiple groups are focused on SOD1 RNA interference-based approaches to remove SOD1. What is clear is that care should be taken not to inadvertently downregulate the protective SOD1 in peripheral Schwann cells. In fact, perhaps efforts to simultaneously downregulate CNS-expressed SOD1 and upregulate Schwann cell SOD1 might be an ideal therapeutic strategy.

References:
Reaume AG, Elliott JL, Hoffman EK, Kowall NW, Ferrante RJ, Siwek DF, Wilcox HM, Flood DG, Beal MF, Brown RH, Scott RW, Snider WD. Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury. Nat Genet. 1996 May;13(1):43-7. Abstract

View all comments by Christine Vande Velde