This paper by C. S. Lobsiger et al. is an experimental and conceptual tour de force. It combines laser capture microdissection (LCM) with Affymetrix oligonucleotide array analysis in superoxide dismutase 1 (SOD1) active versus inactive mutant rodent models of amyotrophic lateral sclerosis (ALS), and it provides potentially breakthrough results in the biology of ALS motor neuron degeneration. Essentially, the group isolated relatively pure populations of lumbar motor neurons from embryonic rat preparations, as well as adult motor neurons in mutant mouse models of SOD1 prior to the appearance of clinical symptoms. Importantly, the group compared embryonic motor neurons that expressed high levels of mutant SOD1 with corresponding wild-type controls overexpressing SOD1 at comparable levels that do not develop ALS-like symptoms. Similarly, adult motor neurons were acquired via LCM from mutant mice expressing dismutase-active SOD1 (the G37R mutant), dismutase-inactive SOD1 (the G85R mutant), wild-type SOD1 overexpressing mice, and non-transgenic littermate controls. The experimental...  Read more

This paper by C. S. Lobsiger et al. is an experimental and conceptual tour de force. It combines laser capture microdissection (LCM) with Affymetrix oligonucleotide array analysis in superoxide dismutase 1 (SOD1) active versus inactive mutant rodent models of amyotrophic lateral sclerosis (ALS), and it provides potentially breakthrough results in the biology of ALS motor neuron degeneration. Essentially, the group isolated relatively pure populations of lumbar motor neurons from embryonic rat preparations, as well as adult motor neurons in mutant mouse models of SOD1 prior to the appearance of clinical symptoms. Importantly, the group compared embryonic motor neurons that expressed high levels of mutant SOD1 with corresponding wild-type controls overexpressing SOD1 at comparable levels that do not develop ALS-like symptoms. Similarly, adult motor neurons were acquired via LCM from mutant mice expressing dismutase-active SOD1 (the G37R mutant), dismutase-inactive SOD1 (the G85R mutant), wild-type SOD1 overexpressing mice, and non-transgenic littermate controls. The experimental design enabled a provocative microarray analysis that was validated at the protein level via immunocytochemistry for several individual candidates.

The study presented three major observations. First, no overt transcriptome-related results were found in the embryonic motor neuron preparations, suggesting that any alterations at this early time point are not the result of SOD1-induced transcriptional alterations in vivo. Second, marked alterations in the motor neuron transcriptome were found prior to the presentation of overt clinical symptoms in the mutant mouse lines, indicating that age-related alterations in gene expression may drive the toxic process well before dysfunction becomes apparent clinically. This finding has tremendous implications for human ALS, and at-risk relatives of families with familial ALS should be investigated from an early age. Moreover, additional single cell/population cell microarray studies of motor neurons obtained postmortem are warranted in ALS cases (both sporadic and familial), even though the disease process may be at the end stages when tissues become available. Third, several common classes of transcripts appear to be involved in the pathogenesis of mutant SOD1 (both dismutase-active and dismutase-inactive forms) motor neuron degeneration. These include genes for the neuronal regenerative/injury response, the complement pathway, and the D/L serine biosynthetic pathway. All three classes are interesting, and relatively novel for the investigation of motor neuron disease within mutant SOD1 models, and are certainly worth extensive experimental follow-up.

Importantly, the LCM-based paradigm enabled the investigators to demonstrate that induction of several classical complement-related genes was fairly neuron-specific, and not a contamination effect from proliferating microglial cells. Previous regional and spinal cord microarray evaluations in mutant SOD1 models were not able to reach this level of single cell/homogeneous population resolution, and consequently were unable to provide such in-depth analysis and interpretation. In summary, this work illustrates the power of combining state-of-the-art functional genomics approaches in well-characterized mutant animal models of neurodegeneration (along with appropriate wild-type overexpressing controls) with microarray analysis for high-throughput evaluation of relevant classes of transcripts that may have functional and/or mechanistic implications for human neurological disorders.

View all comments by Stephen D. Ginsberg

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)...  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