Glia – Absolving Neurons of Motor Neuron Disease

In courtroom dramas, the savvy attorney always manages an acquittal for the innocent defendant. Research dramas can be similar. While the obvious suspects for amyotrophic lateral sclerosis (ALS), aka motor neuron disease, would seem to be the motor neurons, growing evidence suggests that these specialized cells are victims more than perpetrators. In at least one form of ALS—that caused by superoxide dismutase (SOD) mutations—it is becoming increasingly clear that glia are the guilty party. In 2003, Don Cleveland and colleagues at the University of San Diego showed that glial expression of mutant SOD is sufficient to cause ALS-like symptoms in mice (see ARF related news story). Now, working independently, researchers at Harvard University and Columbia University, New York, come to similar conclusions. Their findings, together with some new data from the Cleveland lab, may help uncover how and why glia cause harm to motor neurons and may lead to new therapeutic approaches for ALS and, perhaps, other neurodegenerative diseases.

Writing in the April 15 Nature Neuroscience online, the Harvard researchers, led by Kevin Eggan and Tom Maniatis, reported that motor neurons coaxed from mouse embryonic stem cells grow fairly well when surrounded by normal glia, but degenerate more rapidly when cultured together with glia expressing the SOD G93A mutant that causes ALS. In the same issue of the journal, the Columbia group, led by Serge Przedborski, described a slightly different in-vitro approach. They reported that while expression of mutant SOD in primary motor neurons does not cause neurodegeneration, astrocytes expressing the toxic protein kill both primary neurons and motor neurons derived through stem cell differentiation. Since these researchers found that neurons grow fine when co-cultured with fibroblasts, microglia, and myocytes expressing mutant SOD, they reasoned that some toxic factor specific to astrocytes must mediate the demise of motor neurons. Przedborski and colleagues show that the degenerative process requires expression of Bax, a protein that induces programmed cell death, or apoptosis, suggesting it is or it might be a key mediator of glial-neuron interactions. Cleveland and colleagues identified some other factors that might mediate toxicity. They used DNA arrays to compare gene expression patterns in neurons taken from young and adult mice expressing wild-type and mutant forms of SOD.

Maniatis, Eggan and colleagues isolated embryonic stem cells from SOD G93A mice crossed with mice expressing a green fluorescent reporter that is only expressed in motor neurons. Joint first authors Francesco Di Giorgio and Monica Carrasco were then able to identify differentiated motor neurons by their green fluorescence. Though the numbers of surviving motor neurons dropped dramatically within two weeks of taking cells from embroid bodies, some motor neurons survived as long as 54 days after plating. Overall, the researchers found that only about half as many SOD G93A-positive neurons survived as wild-type. To determine if this poor survival may be related to other cells that co-differentiate in the cell cultures, such as glia, the scientists plated differentiated neurons on primary glial cultures obtained from SOD G93A or wild-type mice. Initially, the motor neurons seemed to thrive in either environment, but by 14 days, all neurons growing on glia harboring mutant SOD had begun to falter. There was a 50 and 32 percent decrease in wild-type and SOD-mutant neurons, respectively, when grown on the toxic compared to wild-type glia.

Findings from the Przedborski lab are strikingly similar. Joint first authors Makiko Nagai, Diane Re, Tetsuya Nagata, and colleagues plated primary motor neurons from 12.5-day-old mouse embryos on either a poly-D-lysine/laminin substrate or on astrocyte monolayers. While the numbers of surviving neurons dropped by about 25 percent over 2 weeks, the loss was independent of SOD genotype. Neurons isolated from transgenic mice expressing several SOD variants that cause ALS, including G93A, G37R, and G85R SOD, survived as well as those from normal mice or transgenic mice expressing normal human SOD. However, it was a different story when the researchers plated neurons on mutant astrocytes.

Compared to those grown on normal astrocytes, the number of primary neurons, either wild-type or expressing SOD G93A, dropped by about half within 7 days of plating onto SOD G93A astrocyte monolayers. All astrocyte mutations (G93A, G37R, and G85R) had a similar detrimental effect on primary neurons and embryonic stem cell-derived motor neurons. Significantly, the combination of both mutant neurons and mutant astrocytes did not exacerbate neuronal losses when compared to wild-type neurons grown on mutant astrocytes. Thus, these data indicate that expression of mutated SOD1 in both astrocytes and motor neurons did not exacerbate the death or the morphometric changes of PMNs caused by its expression in astrocytes alone, write the authors.

How do mutant astrocytes inflict damage on motor neurons It appears they may be releasing a toxic soluble factor. When Nagai and colleagues challenged primary neurons with conditioned astrocyte medium, they found that less than 50 percent survived the first 7 days compared to neurons grown in medium conditioned by normal astrocytes. Medium conditioned by several other SOD G93A cell types, including myocytes, fibroblasts, and microglia, had no effect, suggesting that only astrocytes secrete the soluble factor. The authors were able to extend survival of motor neurons on toxic astrocyte monolayers by incubating the cultures with the Bax antagonist V5 (a VPMLK pentapeptide). This suggests that Bax-induced cell death might be at least partly to blame for the neurodegeneration, at least in vitro. In support of this, the authors found that V5 also counteracted the elevation of neuronal fractin, a fragment of β-actin released during apoptosis.

Analyzing the response of motor neurons to toxic astrocytes is a goal shared by Cleveland and colleagues. As reported in today’s PNAS online, first author Christian Lobsiger and colleagues used laser microdissection to pluck out about 3,000 ventral horn motor neurons from adult mice. The researchers then used gene arrays to compare the expression of 30,000 transcripts in normal and mutant SOD neurons and also the expression profiles of purified embryonic motor neurons.

Lobsiger and colleagues found that only 12 genes appeared to be dysregulated in embryonic neurons harboring mutant SOD. The adult cell analysis painted a different picture, however. In neurons taken at 8 weeks of age while the animals were presymptomatic, only seven genes were dysregulated in G37R mutant neurons compared to wild-type, but in the period between 8 and 15 weeks that number jumped to 108. The authors chose a few of these genes for further analysis. Two, 3-phosphoglycerate dehydrogenase (Phgdh) and phosphoserine phosphatase (Psph), that were elevated at week 8, were further elevated at week 15. Both are involved in serine biosynthesis, as is a third gene, Psat1, which the researchers found was also elevated by week 15. The three cases suggest to the authors that serine biosynthesis may be askew in ALS motor neurons. The finding of elevated levels of Phgdh protein support that idea. Serine, an NMDA receptor co-agonist, could be involved in detrimental excitotoxic actions, the authors suggest.

To get a more general picture of gene dysregulation in ALS, the authors also profiled expression patterns in neurons taken from SOD G85R animals. Unlike G37R, this mutant lacks dismutase activity and causes later disease onset. Lobsiger and colleagues found 21 genes dysregulated in G85R neurons that are common to those identified in the G37R animals. The genes fall into three main groups: neuronal regeneration/injury; the complement system; and the lysosomal degradation machinery (see the paper and supplementary information online for a full list of the dysregulated genes). Again, the authors confirmed some of the transcriptional analysis by immunohistochemistry. Ventral horn motor neurons of mutant animals had elevated levels for ATF3 and Sprr1a, both involved in regeneration/injury responses, and complement C1q. The unexpected induction of mRNAs of the classic complement pathway (C1qa, C1qb, C1qc) long before appearance of obvious clinical symptoms and before major neuroinflammation, suggests that mutant SOD1-induced upregulation of motor neuron-derived complement components is a likely aspect of a toxicity developed within motor neurons that contributes to neurodegeneration, write the authors. C1q has also been implicated in AD pathology (see Fonseca et al., 2004). Whether astrocyte-derived soluble factors cause any of these neuronal transcriptional changes is unclear.

All told, the three papers suggest that glial/neuron interactions may set off a chain of events that specifically arises from astrocytes and is specifically toxic to motor neurons. Nagai and colleagues found that mutant astrocytes had no effect on GABAergic, dorsal root ganglion, or stem cell-derived cortical interneurons. The three papers also identify some likely pathways that might be involved, which could, perhaps, lead to novel therapeutic interventions. The model system described here may also provide a high-throughput cell-based assay for small molecules that promote survival of mutant SOD1 motor neurons, write Eggan and colleagues.

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. Nature Neuroscience. 2007, April 15. Online publication. 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. Nature Neuroscience. 2007, April 15. Online publication. Abstract

Lobsiger CS, Boillee S, Cleveland DW. Toxicity from different SOD1 mutants dysregulates the complement system and the neuronal regenerative response in ALS motor neurons. PNAS. 2007, April 16. Online publication. Abstract

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