© 2002 Nature Publishing Group http://medicine.nature.com
NEWS & VIEWS were disrupted in the brain after birth, progressive mid-life neurodegeneration resulted. The degeneration was confined to the dorsolateral striatum and CA1 and dentate gyrus fields of the hippocampus. These results providing compelling evidence that CREB-family transcription factors have a fundamental role in the survival of neurons in vivo. Mice lacking CREB also exhibit excess death of neurons in the developing peripheral nervous system5. Thus, it is now clear that CREB family members act as general survival factors by controlling the transcription of antideath or cell survival–promoting genes. But how do they do it? The authors ruled out one obvious possibility—that CREB acts through modulating levels of expression of pro-survival and pro-apoptotic members of the Bcl-2 family. Bcl-2 itself is implicated as a CREB target, but the authors found no alterations in expression of Bcl-2 family members in brain neurons lacking both CREB and CREM. In the future, identification of CNS neuron survival factors whose expression is dependent on CREB will provide fresh insights into neuronal survival and maintenance, and potentially could identify novel therapeutic targets for neurodegenerative disorders. A particularly important feature of the new findings is the preferential degeneration of the striatum in mice lacking both CREB and CREM. This presents fascinating parallels with HD, which also leads to the preferential loss of neurons in the striatum. HD is due to the expansion of polyglutamine repeats in the huntingtin gene. Several lines of evidence suggest that expanded polyglutamine repeats within huntingtin interact with and sequester transcriptional cofactors. Remarkably, polyglutamine repeat proteins interact with key effectors of CREB, CBP and P/CAF, as well as the coactivator TAFII130, displacing them from their nor-
mal nuclear locations6–10. The polyglutamine expansion of huntingtin may thus interfere with both CBP and CREB-mediated transcription9,10, resulting in death of cultured neurons9. The CREB effectors CBP and P/CAF have intrinsic acetyltransferase (HAT) activity, which appears to be impaired in polyglutamine disorders. Consistent with the latter notion are observations that enhancing levels of acetylation with deacetylase inhibitors prevents apoptosis of cultured cells caused by nuclear-targeted polyglutamine11 and rescues transgenic flies from the degenerative effects of mutant huntingtin8. CBP and other HATs associate with a large number of transcription factors, so the mechanism by which their sequestration by polyglutamine expansion proteins results in apoptosis is still unclear. Neuronal degeneration may be due to compromised levels of CREB-dependent transcription or diminished activities of other transcription factors. The extraordinary degeneration of striatal neurons in CREB/CREM mutant mice lends support to a model in which a deficiency in CREB signaling accounts for neuronal loss associated with HD. The new findings may also help explain a paradox of HD—projection neurons of the striatum are selectively lost in patients with HD despite the fact that huntingtin is widely expressed in both neuronal and non-neuronal cells. The observation that non-striatal regions of the brain, including the nucleus accumbens, remain largely unaffected by the loss of CREB and CREM may provide a clue. Indeed, many of the same neurons unaffected by CREB/CREM loss are also spared in patients with HD. Perhaps adult striatal neurons are unusually dependent on CREB-dependent transcription for survival, rendering them particularly vulnerable to polyglutamine-
mediated depletion of CREB effectors. Lastly, Schutz and colleagues also provide strong evidence for the idea that excess apoptosis can account for the slow neurodegeneration that occurs in patients with devastating neurodegenerative disorders such as HD. The work of Schutz and colleagues should give direction to future research examining the relationship between transcription and apoptosis in neurodegenerative disorders. 1. Mantamadiotis, T. et al. Disruption of CREB function in brain leads to neurodegeneration. Nature Genet. 31, 47–54 (2002). 2. Mayr, B. & Montminy, M. Transcriptional regulation by the phosphorylation-dependent factor CREB. Nature Rev. Mol. Cell. Biol. 2, 599–609 (2001). 3. Bonni, A. et al. Cell survival promoted by the RasMAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 286, 1358–1362 (1999). 4. Riccio, A., Ahn, S., Davenport, C.M., Blendy, J.A. & Ginty, D.D. Mediation by a CREB family transcription factor of NGF-dependent survival of sympathetic neurons. Science 286, 2358–2361 (1999). 5. Lonze, B.E., Riccio, A., Cohen, S. & Ginty, D.D. Apoptosis, axonal growth defects and degeneration of peripheral neurons in mice lacking CREB. Neuron (in the press). 6. McCampbell, A. et al. CREB-binding protein sequestration by expanded polyglutamine. Hum. Mol. Genet. 9, 2197–2202 (2000). 7. Steffan, J.S. et al. The Huntington’s disease protein interacts with p53 and CREB-binding protein and represses transcription. Proc. Natl. Acad. Sci. USA 97, 6763–6768 (2000). 8. Steffan, J.S. et al. Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature 413, 739–743 (2001). 9. Nucifora, F.C. Jr et al. Interference by huntingtin and atrophin-1 with CBP-mediated transcription leading to cellular toxicity. Science 291, 2423–2428 (2001). 10. Shimohata, T. et al. Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription. Nature Genet. 26, 29–36 (2000). 11. McCampbell, A. et al. Histone deacetylase inhibitors reduce polyglutamine toxicity. Proc. Natl. Acad. Sci. USA 98, 15179–15184 (2001).
Departments of 1Neurology, 2Neuroscience and 3 Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine Baltimore, Maryland, USA Email:
[email protected] or
[email protected]
An array of possibilities for multiple sclerosis Brain lesions exhibiting inflammation and neuronal damage are an important part of the pathology of multiple sclerosis. Microarray analysis compares gene expression in two forms of these lesions, and points to new therapies for the disease. (pages 500–508)
M
ultiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) that afflicts a broad spectrum of people. MS results when T lymphocytes and other immune-system cells infiltrate the white matter of the CNS. The inflammation and subsequent destruction of myelin cause progressive paralysis and other neurological symp-
STEPHEN M. TOMPKINS & STEPHEN D. MILLER toms. These can include muscular tremors, numbness, itching, color-blindness, double vision, loss of vision, loss of coordination or balance, acute paralysis and cognitive impairment. Additionally,
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patients can exhibit chronic symptoms or suffer recurring relapses. This large diversity of symptoms and their variability confound both the diagnosis and understanding of MS. While it is clear that an aberrant immune response against the CNS mediates disease, an understanding of the exact nature of this response has remained elusive. 451
© 2002 Nature Publishing Group http://medicine.nature.com
NEWS & VIEWS In this issue, Lock et al.1 bring us closer to comprehending the mechanisms that contribute to MS. They make significant headway in characterizing the chronic and acute phases of illness using microarray analysis of tissue from neuronal lesions that occur in MS. They compare gene expression in two distinct types of neuronal lesions: acute lesions, characterized by inflammation, and chronic ‘silent’ lesions, which show extensive scarring and demyelination. The authors then go beyond descriptive science and test two identified genes as possible therapeutic targets. Using cytokine therapy or targeted gene disruption, they are able to alleviate symptoms in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). This is the first description supporting molecular differences in the two types of lesions, and provides data that could ultimately influence the choice of treatment for acute versus chronic stages of the disease. Much of our knowledge of MS has derived from the study of EAE, a mouse autoimmune model of MS. EAE is a T cell–mediated autoimmune disease specific for protein components of CNS myelin, resulting in a course of paralysis with clinical and histopathological similarities to MS (ref. 2). This makes it a useful model for studying human autoimmune disease. However, a word of caution: clinical trials have shown that many therapies proven in the mouse have not been efficacious in humans (Fig. 1). In humans, the inability to collect brain or spinal cord biopsies from MS patients has hindered our understanding of the initiation and progression of the disease. Furthermore, a scarcity of post-mortem tissue has impeded analysis of the late stages of MS, as scientists have been unable to garner substantial information from the tissues made available. Now, with the advent of gene microarray technology, we can collect exponentially more information from a single MS lesion tissue sample than was previously possible. Although research based on microarray analysis is highly descriptive, the technique is powerful and has many applications. The work of Lock et al. is a prime example of the value of microarray technology. The authors were able to show pronounced differences in gene expression in tissue obtained post-mortem from four MS patients compared with tissue from two individuals without MS. 452
Acute lesion Gene expression in humans
Mouse studies
Chronic lesion
Human studies
Gene expression in humans
Mouse studies α-VLA-4 treatment inhibits EAE early, but exacerbates late in disease
Human studies Anti-α4 integrin treatment inhibits early lesion development but increases relapse rate in clinical trials.
Rearranged immunoglobulin chains
Intravenous immunoglobulin (IVIG) treatment has therapeutic effect
IVIG treatment alleviates some symptoms.
α4 integrin (VLA-4)
MAP kinases
Upregulated in encephalitogenic T cells
Not tested
Histamine receptor H1
Anti-histamine treatment inhibits disease
Not tested
TGFβ (transforming growth factor β )
TGFβ treatment inhibits disease
TGFβ trial halted due to toxic side effects
IgG and IgE receptors
Ig receptor knockout mice show reduced symptoms
IVIG treatment has therapeutic effect
TNFR p55/p75 knockout & TNFR p55 knockout mice resistant to disease
TNFR blockade exacerbates disease
TNFR p75 knockout mice exhibit enhanced disease
α-TNF treatment exacerbates disease
5- lipoxygenase (5-LO)
5-LO inhibitors suppress EAE
Not tested
G-CSF (granulocyte colony stimulating factor)
G-CSF treatment attenuates disease
Not tested
TNFR (tumor necrosis factor receptor)
Fig. 1 Understanding the differences between silent and acute MS lesions will help tailor therapies for distinct stages and forms of the disease. A number of potential targets suggested by recent gene array data have been tested in both animal disease models and clinical trials with a spectrum of results. The results from the current study are highlighted in light yellow.
They demonstrated that 39 genes had increased expression in all MS lesions, whereas 49 genes had decreased expression. Genes with increased expression included the class II antigen presentation complex, immunoglobulin, complement and numerous pro-inflammatory cytokines. Neuron-associated genes as well as genes associated with myelin production were underexpressed in all lesions. Moreover, acute and silent lesions each showed upregulation of a distinct set of genes (22 and 32 genes, respectively). This is the first description supporting molecular differences in the histologically characterized acute and silent MS lesions. Surprisingly, many of the inflammatory molecules considered important in MS and EAE (for example, interferon-γ, tumor necrosis factor-α, interleukin-12 and others) were not found in either acute or chronic lesions. Whether this will hold true with expanded sampling is uncertain, but this observation certainly warrants further investigation. The power of the microarray approach was fully demonstrated when the authors chose two genes to test as potential therapeutic targets: those encoding the immunoglobulin Fc receptor, which is upregulated in silent lesions, and granulocyte-colony stimulating factor
(G-CSF), which is upregulated in acute MS lesions. To address the requirement of Fc receptors in EAE, the authors compared the severity of disease in Fc receptor–deficient and wild-type mice. They discovered that acute disease was less severe and chronic disease was absent in mice lacking Fc receptor expression. In a second validation trial Lock et al. assessed the capacity of G-CSF to affect the course of EAE. Treatment with G-CSF before the onset of disease decreased the severity of the early stages of the disease but had no effect later. Zavala et al. recently showed decreased disease severity upon G-CSF treatment at the acute phase of disease3. Their work in conjunction with these new results suggest that G-CSF may be expressed early as a mechanism to downregulate acute disease. Other groups have also used gene arrays to measure gene expression in MS lesions as well as in CNS tissues of mice with EAE. Whitney et al. screened acute lesions from a single MS patient for some 5,000 gene transcripts4. In a separate study, the same group compared the expression of roughly 2,800 genes from both MS and EAE CNS tissues5. Ibrahim et al. measured the expression of about 11,000 gene transcripts from the CNS of EAE mice6. These studies represent an enormous amount of data. The focus of
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© 2002 Nature Publishing Group http://medicine.nature.com
NEWS & VIEWS each of these reports is quite different, so although it is highly likely that some of the data are complementary, the comparisons are not presented within the publications. This problem highlights the necessity of developing gene-array databases. Creating a repository of raw gene-array data and providing the tools for analysis would allow scientists to meaningfully compare data from published microarray studies. A resource of this nature would fully harness the potential of gene array technology. Although not unique, the report of Lock et al. is noteworthy for several reasons. The authors show the ideal application of microarray technology and compare acute and silent lesions and find pronounced differences in the tissues. These observations highlight the importance of understanding the distinct stages and different forms of MS. Moreover, the data may enable scientists to tailor therapeutic strategies to stages and/or forms of MS. Additionally, the new study validates data from many EAE
studies and provides direction for new animal research, as shown by the current study as well as a variety of other studies mentioned by Lock et al. as in preparation for publication. Beyond EAE, this work will directly affect studies with MS patients by providing data to help explain clinical trials, as well as initiate new investigations. In the future, we hope to see databases correlating gene-analysis data such as that of Lock et al. with data from clinical trials as well as animal research. The association of all these types information is invaluable to scientists studying MS as well as any other disease. The seedling of such a daunting project can be found at the Stanford Microarray Database (http://genome-www5.stanford.edu/microarray/smd/). Here, microarray data from a variety of diseases and analysis tools are available to the public. If this project grows, it will certainly bear fruit to the benefit of researchers and patients alike, and maximally utilize the data obtained in
the current study and others that are sure to follow. 1. Lock, C. et al. Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nature Med. 8, 500–508 (2002). 2. Wekerle, H., Kojima, K., Lannes-Vieira, J., Lassmann, H. & Linington, C. Animal models. Ann. Neurol. 36 (Suppl.), S47–S53 (1994). 3. Zavala, F. et al. G-CSF therapy of ongoing experimental allergic encephalomyelitis via chemokineand cytokine-based immune deviation. J. Immunol. 168, 2011–2019 (2002). 4. Whitney, L.W. et al. Analysis of gene expression in mutiple sclerosis lesions using cDNA microarrays. Ann. Neurol. 46, 425–428 (1999). 5. Whitney, L.W., Ludwin, S.K., McFarland, H.F. & Biddison, W.E. Microarray analysis of gene expression in multiple sclerosis and EAE identifies 5-lipoxygenase as a component of inflammatory lesions. J. Neuroimmunol. 121, 40–48 (2001). 6. Ibrahim, S.M. et al. Gene expression profiling of the nervous system in murine experimental autoimmune encephalomyelitis. Brain 124, 1927–1938 (2001).
Departments of Microbiology-Immunology and the Interdepartmental Immunobiology Center Northwestern University Medical School, Chicago, Illinois, USA Email:
[email protected]
Corticosteroids and cardioprotection New findings in mice suggest that corticosteroids mediate nitric oxide production in the endothelium, which in turn protects the heart against damage when deprived of oxygen. The mechanism explains, at least in part, the cardioprotective effects of these anti-inflammatory agents. (pages 473–479)
H
eart disease is the leading cause of death worldwide, and its prevalence is rapidly increasing in non-industrialized countries. Myocardial infarction is the irreversible necrosis of heart muscle secondary to prolonged oxygen deprivation; it results in impaired contractile function, increased predisposition to arrhythmias and other long-term complications. In the United States alone, each year approximately 1.5 million people suffer from acute myocardial infarction and 500,000–700,000 die from a coronary artery–related event. Corticosteroids—potent anti-inflammatory and immunosuppressive agents commonly used in the treatment of several disorders—protect the heart from ischemic injury1, but before these compounds can be considered for therapy in ischemic heart disease, a better understanding of their mechanism of action must emerge. In this issue, HafeziMoghadam and colleagues at Harvard Medical School suggest that regulation of endothelial nitric oxide (NO) synthase (eNOS) activity plays a key role in the process2.
CHRISTOPH THIEMERMANN Myocardial infarction and other acute coronary syndromes occur in response to an imbalance of oxygen supply and demand; reperfusion of the previously ischemic myocardium and the associated inflammatory response contribute to the development of cardiac injury. Although anti-inflammatory corticosteroids protect the heart against ischemic injury1, they also reduce wound healing and scar formation, leading to the development of cardiac aneurysms and potentially fatal cardiac ruptures3. The adverse effects of corticosteroids have been attributed to their genomic effects. Corticosteroids bind to nuclear glucocorticoid receptors, which in turn modulate the expression of target genes by binding to DNA sequences containing the glucocorticoid response elements (GRE). Which target genes are involved in the known cardioprotective effects of corticosteroids remains an important question. The study by Hafezi-Moghadam and col-
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leagues reports that high-dose corticosteroids protect the heart and cremaster muscle of the mouse against ischemiareperfusion injury by causing the rapid (within 10–60 min), non-transcriptional activation of eNOS2. Specifically, the authors show that binding of dexamethasone to the glucocorticoid receptor results in the stimulation of phosphatidylinositol 3-kinase and protein kinase Akt, which cause activation of eNOS and enhanced NO formation. Once formed by vascular endothelial cells, NO diffuses to adjacent cells and activates soluble guanylate cyclase (sGC) by binding to the iron on its heme component. Activation of sGC results in a reduction in intracellular calcium concentration, which in turn mediates many (but not all) of the effects of NO, including vasodilatation and inhibition of platelet and neutrophil adhesion (Fig. 1). In wild-type mice, the cardioprotective effects of corticosteroids were abolished by antagonists of the nuclear glucocorticoid receptor and inhibitors of eNOS activitiy (NG-nitroarginine methyl ester)3. Most notably, no cardioprotective effects of corti453
