Microarrays tantalize investigators with the promise of a new smorgasbord of genes to explore and proteins to target with therapeutics. But these studies are so variable, say researchers from the University of California, Los Angeles, David Geffen School of Medicine, that findings rarely overlap even when scientists are looking for genes involved in the same disease. For example, among 11 recent microarray studies for amyotrophic lateral sclerosis, 1,140 known genes have popped up. Yet only 95 were identified in more than one study, and of those, only 61 genes moved in the same direction (up- or downregulated with disease) in multiple studies.

The UCLA researchers, led by Stanislav Karsten and Martina Wiedau-Pazos, set out to conduct an ALS microarray study that would yield more reliable results. They shored up their study with multiple cell types from mice as well as human tissue, and considered different time points in disease progression. Four years later—this kind of careful validation takes time—they report their results in the June 18 Human Molecular Genetics online. Joint first authors Lili Kudo and Liubov Parfenova and their colleagues identified a number of potentially useful genes. Among the list are a baker’s dozen that could serve as blood biomarkers, which are sorely needed in ALS research.

There are several reasons previous studies did not corroborate each others’ results, the authors say. For one, when different scientists use different microarrays, and analyze their data differently, it is no surprise that one study does not match the next, Karsten said. And shortcomings in study design can also confuse results. “One of the main problems is that the brain is a very complex tissue,” Karsten said. “Unfortunately, most of the previous studies neglected this fact and used whole tissue. If we really want to understand the mechanisms of the disease, we have to look at specific cells.” Furthermore, in cases where researchers screen postmortem tissue, they only see gene changes at the end stage of disease, when neurons are already dead or dying.

The UCLA researchers designed their study to overcome some of these challenges. They compared a gene expression in wild-type mice with expression in two mouse models of ALS,. One was a classic model, expressing human mutant superoxide dismutase 1 (SOD1), a common cause for familial ALS. The other was a tau mutant mouse, which is relevant to frontotemporal dementia, a disease that shares much in common with ALS. The researchers collected tissue from these animals in the presymptomatic stage, hoping to identify common genes associated with the cause of the disease, not simply the effects of neural degeneration. And instead of whole tissue, they used laser capture microdissection to collect motor neurons as well as glia, which have been repeatedly linked to ALS pathogenesis.

From their initial screen, the researchers identified 251 genes, including 65 unknowns, that were differentially expressed in one of the ALS models compared to control animals. “We hoped that we would find a lot of genes in common,” Karsten said. “But in fact, these two disease processes are almost entirely different, at least at the level of global transcripts.” Only 12 genes—eight known—were differentially regulated in both the SOD1 and tau models.

To validate the microarray results, the researchers used RT-PCR and immunohistochemistry to confirm the changes in a few select genes. Even for genes that had small, 1.5-fold changes, there was noticeable change in protein expression under the microscope, they report.

Because a genetic mouse model can be a poor substitute for true human disease, the scientists sought to confirm the importance of these genes in human ALS samples. They used postmortem material from people who had sporadic ALS and from normal controls. They tested 10 top candidates, based on their mouse data and the availability of antibodies, and performed immunohistochemistry on the human samples. Among the 10, three genes were upregulated in both SOD1 and tau mouse models and people with ALS: CRB1, CNGA3, and OTUB2. Another, MMP14, was consistently downregulated. Because these genes were regulated similarly in two genetic models and sporadic cases, Wiedau-Pazos said, they may be relevant to sporadic as well as inherited ALS, Expression of the other six genes was inconsistent between mice and people, but Wiedau-Pazos noted this is not too surprising in a comparison of early mouse disease to late human disease.

Finally, the researchers went back to the mice to analyze gene changes in the peripheral blood. For clinical researchers to properly diagnose, assess disease progression, and evaluate drug efficacy, they need easily accessible biomarkers. The UCLA team designed a custom microarray with 1,449 potentially ALS-related genes from their earlier screen, and used it to test blood samples from mSOD1 mice. They were able to detect 13 genes that reliably went up or down in mSOD1 blood, compared to control wild-type blood. Among these were three genes already of interest in ALS. Neurofilament heavy chain and peripherin, both involved in motor neuron cytoskeletal structure, were upregulated in mSOD1 mice. Monoglyceride lipase, which inactivates endogenous cannabinoids and may be protective in the disease, was consistently downregulated in blood from the mSOD1 mice, (Micale et al., 2007).

The next step, the authors said, will be to validate these blood markers in human samples. If it works, Karsten noted, “it would be an incredibly cheap test”—just 13 probes on a chip, plus a drop of blood.

The authors say their approach, with multi-step validation, is the way to get solid results from microarray analysis. Because of that, Wiedau-Pazos thinks the candidates they have identified are likely to be confirmed in future work.—Amber Dance

Comments

Make a Comment

To make a comment you must login or register.

Comments on News and Primary Papers

  1. We have read the very interesting article by Kudo et al. These scientists have performed a careful analysis to identify transcripts (in motor neurons and surrounding cells) that are differentially expressed between control mice and two different mouse models displaying motor neuron degeneration; the mutant SOD1G93A mouse model of familial ALS (fALS) and the mutant TauP301L mouse. They analyzed gene expression differences, using microarrays, in motor neurons, and in cells surrounding the motor neurons, isolated using laser capture microdissection. Their goal was to analyze pre-symptomatic animals in order to avoid studying events downstream of degeneration. The hypothesis was that identification of common molecular targets in the two genetically distinct disease models could represent general markers of neuronal vulnerability, instead of gene-specific effects. Commonly regulated genes could then perhaps translate not only to familiar ALS (fALS), but also to sporadic ALS (sALS).

    Using this strategy, 251 transcripts with altered gene expression were identified. Of these, only 12 transcripts were differentially regulated in both animal models. The microarray data was carefully confirmed using RT-PCR and immunohistochemical analysis. Importantly, of the 12 genes with differential regulation in both animal models, five genes (CNGA3, CRB1, OTUB2, MMP14, and RSPO2) were consistently differentially expressed also in sporadic ALS (sALS) patient material compared to control tissue (as analyzed by custom made tissue microarrays). It would be very interesting to find out if the remaining genes that were not differentially expressed in sALS patient postmortem material could be in fALS patient material. However, if these genes were not regulated similarly in fALS patients as in the disease models, it might reflect discrepancies between the animal models and the human disease(s) or, as suggested by the authors, an issue of comparing pre-symptomatic/early disease in the animal with end-stage of disease in the patients.

    Furthermore, to analyze if any differentially expressed genes could be identified in peripheral blood, and thereby be potential biomarkers of disease, a custom microarray harboring genes that were initially identified as differentially expressed was developed and 13 genes were identified in peripheral blood of the SOD1G93A mice.

    This study shows an example of a very stringent analysis and subsequent confirmation of microarray data on both RNA and protein level. Furthermore, mouse data was compared to sporadic ALS patient data to identify clinical relevance. It will be very interesting to see if the differential gene targets in common between the mutant SOD1 and tau models of motor neuron disease and sALS patient can be used to modulate neuronal vulnerability in vivo. Furthermore, if the markers identified in peripheral blood in the SOD1G93A transgenic mouse can also be inferred to ALS patient material that would be a big step forward in the early diagnosis of ALS (and subsequent future early treatment).

    In the present study, the SOD1G93A mice were analyzed at eight weeks of age in order to analyze pre-symptomatic animals. Paralysis, in general, begins at three months of age in this model of fALS (1,2). It should, however, be noted that the electrical properties of lumbar motor neurons and axonopathy already start during the first and second month of age in this model (3,4), long before onset of symptoms and loss of motor neuron cell bodies. Therefore, the motor neurons that were analyzed in this study were most likely already going through early molecular changes characteristic of their forthcoming demise. Perhaps a complementary analysis of even younger animals, four weeks old, could provide more common targets genes between the two disease models.

    Data from fALS models indicate that factors intrinsic to motor neurons are crucial for initiation of degeneration, while non-cell-autonomous events are instrumental for disease progression (1-9). A careful gene expression analysis of motor neurons and surrounding cells could therefore give clues to intrinsic and extrinsic mechanisms of motor neuron degeneration in ALS. In the present study, the isolation of cell types that were subsequently analyzed by microarrays was based on staining of spinal cord tissue with Cresyl violet. Motor neurons were clearly identified based on size, while punctuate staining of Cresyl violet was used for collection of surrounding cells, defined as glial cells. However, the surrounding cells isolated will be a mixture of astrocytes, oligodendrocytes, microglial cells, and small interneurons that surround (and innervate) motor neurons in the ventral horn. While it has been demonstrated that astrocytes and microglia can drive disease progression in mutant SOD1 mouse models of fALS (1,4), it is still unknown what role interneurons might play (9). While, oligodendrocytes don’t appear to drive initiation of disease in fALS models (10), the specific role of these cells in disease progression remains to be further characterized. In the present study, the microarray analysis of the likely mixture of cells isolated might be less informative than a selective analysis of the individual glial cell types (astrocytes versus microglial cells versus oligodendrocytes) and neuronal populations surrounding (and influencing) motor neurons.

    We have also aimed to identify targets of neuronal vulnerability, but using a different strategy (Hedlund et al., in press). Based on the differential loss of specific motor neuron subpopulations in motor neuron diseases, we isolated individual motor neurons from the oculomotor/trochlear complex (these do not degenerate in ALS), hypoglossal nucleus (show vulnerability in ALS), and from the ventral horn of the cervical spinal cord (degenerate in ALS) using laser capture microdissection in wild-type rats. We hypothesized that dissecting the intrinsic molecular code underlying the normal physiology of motor neurons that display differential vulnerability to disease could provide a basis for revealing why one motor neuron subpopulation is more vulnerable to degeneration than another. Our findings also support the use of gene profiling of vulnerable versus resistant cell populations to understand which molecules and pathways can be modified to protect against disease processes in vivo.

    References:

    . Onset and progression in inherited ALS determined by motor neurons and microglia. Science. 2006 Jun 2;312(5778):1389-92. PubMed.

    . 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.

    . Recovery from paralysis in adult rats using embryonic stem cells. Ann Neurol. 2006 Jul;60(1):32-44. PubMed.

    . Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis. Nat Neurosci. 2008 Mar;11(3):251-3. PubMed.

    . Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons. Nat Neurosci. 2007 May;10(5):615-22. PubMed.

    . 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.

    . Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation. Cell Stem Cell. 2008 Dec 4;3(6):637-48. PubMed.

    . Non-cell-autonomous effect of human SOD1 G37R astrocytes on motor neurons derived from human embryonic stem cells. Cell Stem Cell. 2008 Dec 4;3(6):649-57. PubMed.

    . ALS model glia can mediate toxicity to motor neurons derived from human embryonic stem cells. Cell Stem Cell. 2008 Dec 4;3(6):575-6. PubMed.

    . Mutant SOD1 in cell types other than motor neurons and oligodendrocytes accelerates onset of disease in ALS mice. Proc Natl Acad Sci U S A. 2008 May 27;105(21):7594-9. PubMed.

    View all comments by Eva Hedlund

References

Paper Citations

  1. . Endocannabinoids and neurodegenerative diseases. Pharmacol Res. 2007 Nov;56(5):382-92. PubMed.

Further Reading

Papers

  1. . Biomarkers in amyotrophic lateral sclerosis. Lancet Neurol. 2009 Jan;8(1):94-109. PubMed.
  2. . Gene expression analysis of the murine model of amyotrophic lateral sclerosis: studies of the Leu126delTT mutation in SOD1. Brain Res. 2007 Jul 30;1160:1-10. PubMed.
  3. . Differential expression of genes in amyotrophic lateral sclerosis revealed by profiling the post mortem cortex. Amyotroph Lateral Scler. 2006 Dec;7(4):201-10. PubMed.
  4. . Differential expression of 14 genes in amyotrophic lateral sclerosis spinal cord detected using gridded cDNA arrays. J Neurochem. 2001 Apr;77(1):132-45. PubMed.
  5. . Gene expression profile of spinal motor neurons in sporadic amyotrophic lateral sclerosis. Ann Neurol. 2005 Feb;57(2):236-51. PubMed.
  6. . Molecular signature of late-stage human ALS revealed by expression profiling of postmortem spinal cord gray matter. Physiol Genomics. 2004 Jan 15;16(2):229-39. PubMed.
  7. . Disease mechanisms revealed by transcription profiling in SOD1-G93A transgenic mouse spinal cord. Ann Neurol. 2001 Dec;50(6):730-40. PubMed.

Primary Papers

  1. . Integrative gene-tissue microarray-based approach for identification of human disease biomarkers: application to amyotrophic lateral sclerosis. Hum Mol Genet. 2010 Aug 15;19(16):3233-53. PubMed.