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New Gene for ALS: RNA Regulation May Be Common Culprit
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27 February 2009. Scientists have uncovered a fresh clue in the mystery that is amyotrophic lateral sclerosis. Unexpectedly, it comes from a gene first implicated in cancer, called fused in sarcoma (FUS). In back-to-back Science papers published today, researchers from Massachusetts General Hospital in Boston, and King’s College in London, U.K., report that 5 percent of people with familial ALS harbor a mutation in FUS. Its protein functions in RNA transcription, splicing and trafficking, and it is strikingly similar to the separate ALS-linked protein TDP-43. The parallels between the two proteins suggest to many scientists that ALS may be a disease of RNA mismanagement.
“This is the beginning of a new era, in which we need to look at RNA processing as a potential early pathogenic event in motor neuron death,” said Stanley Appel of the Methodist Hospital System in Houston, Texas, who was not involved in either study.
The U.S. and U.K. groups, led by Robert Brown and Christopher Shaw, respectively, have been collaborating in the hunt for this particular gene for the better part of a decade. Their combined data, from single nucleotide polymorphism analysis of two families with multiple cases of ALS, pointed to a mutation in chromosome 16. Thomas Kwiatkowski, first author on the U.S. paper, found a third family that allowed him to narrow down the locus and discover the FUS mutation. In London, Caroline Vance, joint first author along with Boris Rogelj, carried out the first in-vitro analysis that showed mutant FUS exits the nucleus to form abnormal aggregates in the cytoplasm of cultured cells.
This research took so long, in part, because false leads led the researchers down the wrong path before they were able to focus in on the FUS-containing locus. The target regions that SNP analysis identified in the first two families overlapped by 40 megabases (Ruddy et al., 2003; Sapp et al., 2003). That area was too large to sequence all the candidate genes. When Shaw and another collaborator each found another family with likely mutations in chromosome 16, they pooled the data to identify a much smaller region of overlap. But sequencing every gene in that region found no mutations, and the research dead-ended. Later, additional family members and further calculations showed that the third and fourth families’ mutations did not map to chromosome 16 after all, and the researchers were left with the impossibly long stretch of chromosome with which they started.
The break came when two sisters, both in the early stages of ALS, came to consult Kwiatkowski. They mentioned that their mother had had a similar condition, and that a third sister was developing muscle weakness. With 12 siblings, this Rhode Island family, originally from a small Cape Verde island off the western coast of Africa, provided a wealth of additional genetic samples. “This family showed a possible address which was exquisitely located on top of the chromosome 16 locus,” said Brown, who now works at the University of Massachusetts in Worchester. With the new data, the scientists were able to narrow their target region to four megabases, a manageable stretch of DNA.
Kwiatkowski identified 56 potentially relevant genes in that area. He included FUS, as a gene involved in genome stability, but did not consider it a likely candidate. The task of sequencing FUS, along with other less promising genes, fell to an undergraduate from Smith College, in Northampton, Massachusetts, who was working in the laboratory over the summer. The student, Alexandra Davis, returned to school before Kwiatkowski had time to analyze her results. When he finally looked at FUS, Kwiatkowski recalls, “I said, holy smokes, this is it!” After confirming his findings, he contacted Shaw’s lab, and the two groups sequenced the gene in their two families with chromosome 16 mutations, and in other people with familial ALS.
The Massachusetts group sequenced FUS in nearly 300 unrelated familial ALS cases, and the British scientists sequenced nearly 200 separate cases. All told, approximately 5 percent of people with familial ALS had a FUS mutation, Kwiatkowski said. Among people with familial ALS, approximately 20 percent have a mutation in superoxide dismutase 1, and an additional 5 percent have mutations in TARDBP, which encodes TDP-43. Other genes have also been linked to the disease, but only in a handful of cases.
The scientists found 15 different FUS mutations, mostly clustered in the carboxyl-terminal region of the protein. With rare exceptions, the mutant allele was dominant. Nearly 300 sporadic ALS cases contained no FUS mutations, the Brown group found. The two research groups sequenced FUS in nearly 2,000 control subjects, and found only one instance of a FUS mutation, in a person of Cape Verdean ancestry. “When you have back-to-back reports from different parts of the world and they come up with extremely similar findings…then you know for sure that it’s real,” Appel said.
FUS, also known as TLS (translated in liposarcoma), has many functions in healthy cells. It mends DNA breaks and binds to RNA to participate in transcription, splicing, and transporting mRNA out of the nucleus (reviewed by Law et al., 2006). None of these functions suggest an obvious connection to motor neuron viability. “No one, a priori, would have ever anticipated that it was related to ALS,” Appel said.
FUS protein primarily resides the nucleus, although it also transports mRNAs to other cellular destinations. Vance expressed mutant and wild-type forms of FUS in fibroblast and neuroblastoma cells, but did not expect to see a difference between the two. That’s because when she performed a similar experiment with TDP-43 mutants, their localization mirrored that of the wild-type. But the FUS mutant clearly shifted from the nucleus to the cytoplasm. Vance was so surprised, she recalls, that she asked a lab mate to confirm what she saw. Furthermore, FUS formed aggregates in the cytoplasm of motor neurons, the Shaw group discovered in postmortem tissue samples from three people who had FUS mutations.
What’s Next in FUS Research?
Some 90 percent of people with ALS have a sporadic form that is not inherited. Even so, the data about FUS, TDP-43, and SOD1 mutations may ultimately help scientists solve sporadic ALS as well. “Anytime we learn any other clue for ALS, it’s good for the field,” said Merit Cudkowicz, a neurologist specializing in ALS who is also at Massachusetts General Hospital but was not involved in the current research. Although the FUS mutation does not apply to many of her patients, she said, “it seems that it fits into a general theme of pathways that could be abnormal in ALS.” The 1993 discovery of SOD1 mutations (Rosen et al., 1993), Cudkowicz noted, led to an explosion of research on ALS, with new rodent models and new therapies to try. The recent TDP-43 and current FUS discoveries will likely do the same, she said, recruiting people who study those genes to consider ALS.
Next, Kwiatkowski and colleagues hope to develop model systems based on FUS mutations. A new mutant mouse would be a welcome addition for a field frustrated by drug candidates that look promising in mSOD1 mice but later fail in human trials. Cell culture models, Kwiatkowski said, will also provide an important method to screen for potential drugs.
In addition, Kwiatkowski plans to address the biological mechanism of FUS mutations. Any of its functions, when disrupted, might somehow lead to disease. Alternatively, one of FUS’s RNA targets could be required for motor neuron health. The FUS aggregates seen in motor neurons could be toxic, or could simply be a byproduct of the cell’s attempts to deal with the rogue protein.
At this point, it is not yet certain if FUS mutations reflect a loss or gain of function. “I think you’ve got to go with gain of function; it’s an autosomal-dominant disease,” Vance said. But the cell biology does not seem to follow that standard genetic rule. “It is a loss of function, because [FUS] suddenly goes from the nucleus to the cytoplasm,” suggested Lucie Bruijn, senior vice president for research and development at the ALS Association, headquartered in Calabasas Hills, California, which provided funding for the current studies. Mislocalized, the protein might not get its job done.
Compounding this question is the finding that the FUS mutation in the Cape Verde family that cracked the case is recessive. The parents came from the same village, and the maternal grandparents were first cousins. The three sisters who have the disease are homozygous for the mutation, whereas three elderly aunts and the father, who are heterozygous, show no symptoms. The Cape Verde mutation also causes atypical disease. The mother lived for 14 years with her condition and eventually died of a heart attack, not the respiratory failure that usually ends the life of people with ALS. Typically, a person with ALS is unlikely to live more than a few years after diagnosis. The presence of both dominant and recessive FUS mutations confounds any simple explanation. “We’ve got both, so I’ve been scratching my head about this,” Kwiatkowski said. “Maybe the different mutations disturb different functions.”
FUS Fits into Bigger RNA Picture
RNA-shuttling proteins have been implicated in other conditions. FUS itself forms a part of the abnormal huntingtin-containing aggregates in Huntington disease, although the significance of its presence there is uncertain (Doi et al., 2008). The Fragile X Mental Retardation Protein (FMRP) associates with RNA (Khandjian et al., 2004), and methyl-CpG binding protein 2 (MeCP2), which is associated with Rett syndrome, is involved in RNA splicing (Young et al., 2005). And in ALS, Brown and colleagues recently found that the elongator protein 3 (ELP3) gene, which encodes part of RNA polymerase II, is associated with disease (Simpson et al., 2009). Then, of course, there is TDP-43. “This raises the possibility that there’s something fundamentally important about RNA metabolism that is important for the viability of the neuron,” Brown said.
At the same time, there are differences in the pathology between FUS mutations and TDP-43 proteinopathies. The cells of people with FUS mutations do not contain aggregated TDP-43, as do the majority of sporadic ALS cases, although they do have ubiquitin inclusions in the nucleus. Those features “suggest the mechanisms may be distinct,” wrote Robert Bowser of the University of Pittsburgh, Pennsylvania, in an e-mail to ARF (see full comment below).
The current results mean that ALS scientists will carefully scrutinize the role of RNA management, in familial as well as sporadic forms of the disease. Looking for FUS pathology in sporadic ALS cases is high on Brown’s to-do list, he said. And two-thirds of familial cases still remain unexplained, raising the question of whether more RNA regulators are waiting to be discovered. The search goes on: loci on chromosomes 9 and 20 have been linked to familial ALS, and may be next to give up their secrets.—Amber Dance.
References:
Kwiatkowski TJ Jr, Bosco DA, LeClerc AL, Tamrazian E, Vanderburg CR, Russ C, Davis A, Gilchrist J, Kasarskis EJ, Munsat T, Valdmanis P, Rouleau GA, Hosler BA, Cortelli P, de Jong PJ, Yoshinaga Y, Haines JL, Pericak-Vance MA, Yan J, Ticozzi N, Siddique T, McKenna-Yasek D, Sapp PC, Horvitz HR, Landers JE, Brown RH Jr. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 27 Feb 2009 323:1205-1208. Abstract
Vance C, Rogelj B, Hortobágyi T, De Vos KJ, Nishimura AL, Sreedharan J, Hu X, Smith B, Ruddy D, Wright P, Ganesalingam J, Williams KL, Tripathi V, Al-Saraj S, Al-Chalabi A, Leigh PN, Blair IP, Nicholson G, de Belleroche J, Gallo J-M, Miller CC, Shaw CE. Mutations in FUS, and RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 27 Feb 2009 323:1208-1211. Abstract
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Comments on News and Primary Papers |
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Comment by: Robert Bowser
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Submitted 27 February 2009
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Posted 27 February 2009
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These papers represent exciting work describing a new genetic mutation associated with familial ALS. The results further highlight the importance for RNA processing in at least familial forms of motor neuron disease. Much work remains to determine the exact mechanisms by which FUS modulates motor neuron survival. It may be related to that of TDP-43. However, the lack of cytoplasmic aggregation of TDP-43, and rare ubiquitin inclusions in the patients with FUS mutations, suggest the mechanisms may be distinct. It is interesting that FUS protein did not accumulate in the cytoplasm of motor neurons in sporadic ALS patients, again suggestive that the pathogenic mechanisms of mutant FUS-induced motor neuron degeneration may be distinct from that in sporadic ALS.  View all comments by Robert Bowser |
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Comment by: Eric Frank
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Submitted 27 February 2009
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Posted 27 February 2009
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These studies raise interesting questions about whether one problem in ALS and perhaps other neurodegenerative diseases is that RNA trafficking proteins fail to properly deliver RNAs to dendritic spines. The paper by Kwiatkowski et al. reports evidence that wild-type FUS and TDP-43 may be involved in transporting RNA into dendrites, where it mediates local protein synthesis that can be stimulated by neural activity. The clumping of the mutant form described by both new papers could therefore perturb the transport of RNA. Local protein synthesis in dendrites plays a major role in the activity-dependent modulation of synaptic strength. Changes in synaptic activity have been recently reported in the mouse model of SOD1 mutation ( van Zundert et al., 2008), so it will be worthwhile to examine this issue in the FUS mice that will certainly be developed by these investigators.
View all comments by Eric Frank |
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Comment by: Jeffrey D. Rothstein
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Submitted 2 March 2009
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Posted 2 March 2009
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This is an extremely exiting story in the understanding of ALS pathogenesis. It actually it dates back to 1998—with the first description of mRNA processing errors in sporadic ALS ( Lin et al., 1998), which, interestingly, was made not in the SOD1 mouse model. At the same time, the spinal muscular atrophy gene was discovered. SMA is not unlike a childhood ALS, though predominately lower motor neurons are affected in that disease. The SMA gene defect is involved in RNA metabolism. So for the next 10 years, the SMA field has investigated the pathobiology of the defective protein. At the time it made the link between sporadic ALS and the SMA story intriguing. But there was no clear genetic link (or cause for the changes in sporadic ALS).
Feed forward to 2008, when Chris Shaw and others found a true genetic defect in RNA metabolism-based protein TDP-43. (Of course more work needs to be done on that.) And now another gene by the Shaw group, and now verified by the group in Boston, does set a string of targets that all focus on RNA...
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This is an extremely exiting story in the understanding of ALS pathogenesis. It actually it dates back to 1998—with the first description of mRNA processing errors in sporadic ALS ( Lin et al., 1998), which, interestingly, was made not in the SOD1 mouse model. At the same time, the spinal muscular atrophy gene was discovered. SMA is not unlike a childhood ALS, though predominately lower motor neurons are affected in that disease. The SMA gene defect is involved in RNA metabolism. So for the next 10 years, the SMA field has investigated the pathobiology of the defective protein. At the time it made the link between sporadic ALS and the SMA story intriguing. But there was no clear genetic link (or cause for the changes in sporadic ALS).
Feed forward to 2008, when Chris Shaw and others found a true genetic defect in RNA metabolism-based protein TDP-43. (Of course more work needs to be done on that.) And now another gene by the Shaw group, and now verified by the group in Boston, does set a string of targets that all focus on RNA metabolism and (lower) motor neurons.
By the way, all these cases appear to predominately involve a lower motor neuron form of ALS. The hint from genetics does suggest more of a loss of function rather than gain, but cell biology will ultimately sort that out. We certainly await the generation of mouse or fly models, which are now well underway for TDP-43. However, this may be a particularly difficult target for specific, non-toxic drug therapy.
View all comments by Jeffrey D. Rothstein |
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Comment by: P. Hande Ozdinler
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Submitted 17 March 2009
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Posted 17 March 2009
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These back-to-back papers on the identification of FUS (fused in sarcoma) gene as a new genetic component of ALS open a new era of research and direct our attention to mRNA biology with respect to disease. After the first identification of mRNA processing errors in ALS patients (Lin, Bristol et al., 1998), the discovery of TDP-43 (Neumann, Sampathu et al., 2006) and now the FUS gene clearly indicate the importance of mRNA management in neurodegenerative diseases. Defects in RNA transcription, splicing, and trafficking may be the reason for cell-type-specific degeneration of motor neurons in ALS. Motor neurons both in the cortex and spinal cord are very large excitatory neurons that extend long axons to their targets and require high levels of energy and protein integrity for survival and function. Defects in transcriptional mechanisms may result in splicing defects, which could give rise to formation of non-functional proteins that would deplete the pool of required proteins for cellular function, and these non-functional proteins may form aggregates that are toxic to neurons. In...
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These back-to-back papers on the identification of FUS (fused in sarcoma) gene as a new genetic component of ALS open a new era of research and direct our attention to mRNA biology with respect to disease. After the first identification of mRNA processing errors in ALS patients (Lin, Bristol et al., 1998), the discovery of TDP-43 (Neumann, Sampathu et al., 2006) and now the FUS gene clearly indicate the importance of mRNA management in neurodegenerative diseases. Defects in RNA transcription, splicing, and trafficking may be the reason for cell-type-specific degeneration of motor neurons in ALS. Motor neurons both in the cortex and spinal cord are very large excitatory neurons that extend long axons to their targets and require high levels of energy and protein integrity for survival and function. Defects in transcriptional mechanisms may result in splicing defects, which could give rise to formation of non-functional proteins that would deplete the pool of required proteins for cellular function, and these non-functional proteins may form aggregates that are toxic to neurons. In addition, defects in the trafficking of mRNA may lead to depletion of key proteins that are in high demand locally for motor neuron function. But if FUS has a general function in mRNA transcription, splicing, and trafficking, why do mutations in this gene cause ALS and not other neurodegenerative diseases? What makes motor neurons more vulnerable in the presence of defective FUS? It could be true that in motor neurons FUS controls the transcription of a distinct set of mRNA that is expressed in a cell-type-specific manner in motor neurons, or that FUS controls the production of a key protein that is highly required in motor neurons when compared to other cell-types, and thus motor neurons may become vulnerable first. FUS seems to be the tip of the iceberg. Finding effectors, binding partners including mRNA, may lead to the identification of key components of both familial and sporadic ALS. More work is on the way!
References: Kneussel M. Dynamic regulation of GABA(A) receptors at synaptic sites. Brain Res Brain Res Rev. 2002 Jun ;39(1):74-83. Abstract
Lin CL, Bristol LA, Jin L, Dykes-Hoberg M, Crawford T, Clawson L, Rothstein JD. Aberrant RNA processing in a neurodegenerative disease: the cause for absent EAAT2, a glutamate transporter, in amyotrophic lateral sclerosis. Neuron. 1998 Mar;20(3):589-602. 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
Vance C, Rogelj B, Hortobágyi T, De Vos KJ, Nishimura AL, Sreedharan J, Hu X, Smith B, Ruddy D, Wright P, Ganesalingam J, Williams KL, Tripathi V, Al-Saraj S, Al-Chalabi A, Leigh PN, Blair IP, Nicholson G, de Belleroche J, Gallo JM, Miller CC, Shaw CE. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009 Feb 27;323(5918):1208-11. Abstract
View all comments by P. Hande Ozdinler |
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Comments on Related Papers |
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Related Paper: TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis.
Comment by: George Perry (Disclosure)
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Submitted 12 March 2008
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Posted 21 March 2008
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 I recommend this paper
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Comments on Related News |
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Related News: Less VAPid Now: Role for ALS Protein Gets Substance
Comment by: Giuseppa Pennetta
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Submitted 26 June 2008
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Posted 26 June 2008
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VAPs (VAMP/synaptobrevin associated proteins) are evolutionarily conserved proteins comprising an amino-terminal domain with significant homology to the major sperm proteins (MSPs), a central coiled-coil domain, and a membrane anchor at the carboxy-terminal domain. MSPs are the most abundant proteins in the amoeboid nematode sperm, where they perform both cytoskeletal and signaling functions. In C. elegans, MSPs signal by antagonizing ephrin/Eph receptor pathway to promote oocyte meiotic maturation, ovarian sheath cell contraction, and oocyte microtubule reorganization. In 2004, Nishimura et al. reported a mutation substituting a conserved proline with a serine in a Brazilian family affected by a heterogenous group of motor neuron diseases ranging from amyotrophic lateral sclerosis (ALS) to atypical ALS and spinal muscular atrophy (1). In Drosophila, dVAP modulates number and size of boutons at neuromuscular junctions (2). Loss of function in dVAP disrupts microtubule cytoskeleton and causes an increase in miniature excitatory post-synaptic potentials that...
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VAPs (VAMP/synaptobrevin associated proteins) are evolutionarily conserved proteins comprising an amino-terminal domain with significant homology to the major sperm proteins (MSPs), a central coiled-coil domain, and a membrane anchor at the carboxy-terminal domain. MSPs are the most abundant proteins in the amoeboid nematode sperm, where they perform both cytoskeletal and signaling functions. In C. elegans, MSPs signal by antagonizing ephrin/Eph receptor pathway to promote oocyte meiotic maturation, ovarian sheath cell contraction, and oocyte microtubule reorganization. In 2004, Nishimura et al. reported a mutation substituting a conserved proline with a serine in a Brazilian family affected by a heterogenous group of motor neuron diseases ranging from amyotrophic lateral sclerosis (ALS) to atypical ALS and spinal muscular atrophy (1). In Drosophila, dVAP modulates number and size of boutons at neuromuscular junctions (2). Loss of function in dVAP disrupts microtubule cytoskeleton and causes an increase in miniature excitatory post-synaptic potentials that correlates with an increase in post-synaptic glutamate receptor clustering. It has also been shown that hVAPB, the causative gene of ALS8, rescues the lethality and the neuromuscular junction phenotype associated with loss of DVAP, clearly indicating that the fly protein and human VAP perform homologous functions (3).
Recently, reports from two independent labs (Tsuda et al. and Ratnaparkhi et al.) have provided new and exciting insight on the normal function of VAP proteins and their possible role in the pathogenesis of VAP-induced ALS. Comments on these papers can be summarized as follows.
The paper by Tsuda et al. reports that VAP proteins are cleaved, and an N-terminal fragment of a size compatible with the size of the MSP domain is secreted and binds to the Eph receptors. The pathogenic allele induces the accumulation of the mutant and the wild-type (wt) protein into the ER and a failure to secrete the cleaved MSP domain. Non cell-autonomous effects of the mutant and wt proteins have been reported both at the level of the Drosophila nervous system and the nematode reproductive system. The ability of dVAP to be cleaved and secreted has been shown with an elegant experiment in which the expression of dVAP has been driven in a subset of cells in the wing imaginal discs. A diffusion of dVAP MSP beyond the protein expressing cells was observed. However, there is no direct evidence that this process of cleavage and secretion of VAP proteins is occurring in neurons, in muscles, or in any other tissue that would be more relevant to the human disease.
The ability of the pathogenic allele to induce the formation of aggregates has been previously reported in cell culture (1,4,5) and Drosophila model systems (3). Tsuda and colleagues report that expression of the mutant protein in a null background induces the formation of detergent-insoluble aggregates. Despite the mutant allele being inherited in a dominant manner in humans, these data lead to the important conclusion that the wild-type protein is not necessary for the formation of aggregates. However, an intriguing question arises: how can the presence of these aggregates be reconciled with the ability of the mutant protein to rescue the phenotypes associated with null mutations in dVAP as shown by three independent studies (3, Ratnaparkhi et al., Tsuda et al.). Are these aggregates different from the ones observed when the mutant protein is expressed in the presence of the wt protein?
Other outstanding questions will need to be addressed: which is the protease or proteases responsible for the cleavage? Is the secretion of the MSP domain of VAP proteins occurring through an unconventional mechanism as already proposed for the MSP proteins in C. elegans? Which is the subcellular compartment in which the cleavage occurs?
The paper by Ratnaparkhi et al. focuses on another important aspect, which is the determination of the disease mechanism. In humans, the pathogenic mutation is inherited in a dominant manner. Dominant mutations are due to a gain of function (hypermorphs and neomorphs), dominant-negative interactions (antimorphs) or haplo-insufficiency. Understanding the patho-mechanism of the disease is important as it can indicate new possible strategies for therapeutic interventions. Several lines of evidence support a possible dominant-negative effect of the pathogenic allele. The formation of aggregates, the depletion of the wild-type protein from its normal localization (3,4,5), and the sequestration of the wt protein in the aggregates clearly suggest a dominant-negative effect (4). Moreover, the fact that the pathogenic allele acts as a dominant-negative can be proven if the overexpression of the mutant protein in the presence of the wt protein leads to a phenotype similar to the loss-of-function mutation. Indeed, it has been reported that transgenic expression of the mutant protein induces a reduction in number of boutons (3), a disruption of the presynaptic cytoskeleton (Ratnaparkhi et al.) and a reduction in miniature excitatory post-synaptic potentials (Tsuda et al.). Ratnaparkhi et al. attempt to further support this statement by performing a systematic analysis of mutant phenotypes in different functional contexts. They compared the effect of overexpressing the wt protein with the overexpression of the mutant protein in transgenic lines expressing comparable amounts of transgenes. The expression levels of the proteins were estimated only for the full-length VAP. Although the mutant allele impairs the secretion of the MSP domain, the cleaved product is still produced as shown in several Western blots reported by Tsuda et al. The same Western blots suggest that the levels of the full-length protein and the cleaved MSP domain are not stoichiometrically similar; therefore, restricting the analysis to the expression levels of the full-length protein may be misleading. A cleaved, non-secreted MSP domain could still be responsible for the intracellular, cell-autonomous effects of the protein.
Although there are several lines of evidence supporting a possible dominant-negative effect, there is other evidence suggesting different mechanisms for the disease. Mutant VAP proteins still retain some functional properties of the wt protein such as the ability to self-oligomerize (3,4) and the ability to rescue, at least in part, the mutant phenotype due to the loss of the endogenous protein. The mutant allele has also acquired new functional properties that are not shared by the normal version of the protein such as the propensity to form aggregates and the “floating active zones” phenotype reported by Ratnaparkhi et al. In one report it has also been shown that the mutant protein has an increased ability of inhibiting the activity of ATF6, a transcription factor involved in UPR (6).
We propose that the mutant allele may cause the disease by a combination of mechanisms that include dominant-negative interactions and toxic effects due to gain of new functions.
Although a lot still remains to be done, studies published over the last six months have convincingly shown that the variety of genetic tools available in Drosophila can now be exploited to foster our understanding of the patho-mechanisms responsible for motor neuron diseases in humans.
References:
1. Nishimura AL, Mitne-Neto M, Silva HC, Richieri-Costa A, Middleton S, Cascio D, Kok F, Oliveira JR, Gillingwater T, Webb J, Skehel P, Zatz M. A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am J Hum Genet. 2004 Nov;75(5):822-31. Abstract
2. Pennetta G, Hiesinger PR, Fabian-Fine R, Meinertzhagen IA, Bellen HJ. Drosophila VAP-33A directs bouton formation at neuromuscular junctions in a dosage-dependent manner. Neuron. 2002 Jul 18;35(2):291-306. Abstract
3. Chai A, Withers J, Koh YH, Parry K, Bao H, Zhang B, Budnik V, Pennetta G. hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction. Hum Mol Genet. 2008 Jan 15;17(2):266-80. Abstract
4. Kanekura K, Nishimoto I, Aiso S, Matsuoka M. Characterization of amyotrophic lateral sclerosis-linked P56S mutation of vesicle-associated membrane protein-associated protein B (VAPB/ALS8). J Biol Chem. 2006 Oct 6;281(40):30223-33. Abstract
5. Teuling E, Ahmed S, Haasdijk E, Demmers J, Steinmetz MO, Akhmanova A, Jaarsma D, Hoogenraad CC. Motor neuron disease-associated mutant vesicle-associated membrane protein-associated protein (VAP) B recruits wild-type VAPs into endoplasmic reticulum-derived tubular aggregates. J Neurosci. 2007 Sep 5;27(36):9801-15. Abstract
6. Gkogkas C, Middleton S, Kremer AM, Wardrope C, Hannah M, Gillingwater TH, Skehel P. VAPB interacts with and modulates the activity of ATF6. Hum Mol Genet. 2008 Jun 1;17(11):1517-26. Abstract
View all comments by Giuseppa Pennetta |
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Related News: Less VAPid Now: Role for ALS Protein Gets Substance
Comment by: John Landers
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Submitted 15 July 2008
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Posted 15 July 2008
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I recommend the Primary Papers
Amyotrophic lateral sclerosis is an age-dependent, degenerative disorder of motor neurons that typically develops in the sixth decade and is uniformly fatal, usually within five years. About 10 percent of ALS cases are familial; 20 percent of these are caused by mutations in the gene encoding copper/zinc superoxide dismutase 1 (SOD1). More recently, it has been shown that mutations in the TDP-43 gene are also causative for familial ALS (1-3). The VAPB P56S mutation was originally observed in a large Brazilian family of Portuguese descent that displayed a pattern of dominantly inherited ALS/motor neuron disease across four generations (4). Subsequent studies identified the mutation in at least seven different families, all of Portuguese-Brazilian origin, each displaying a different clinical course ranging from late-onset spinal muscular atrophy (SMA) to typical and atypical ALS (4). Our previous work identified only a single case of a VAPB mutation (P56S) in a screen of 80 familial ALS samples, demonstrating that VAPB mutations are extremely rare (5). As such, why is it important...
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Amyotrophic lateral sclerosis is an age-dependent, degenerative disorder of motor neurons that typically develops in the sixth decade and is uniformly fatal, usually within five years. About 10 percent of ALS cases are familial; 20 percent of these are caused by mutations in the gene encoding copper/zinc superoxide dismutase 1 (SOD1). More recently, it has been shown that mutations in the TDP-43 gene are also causative for familial ALS (1-3). The VAPB P56S mutation was originally observed in a large Brazilian family of Portuguese descent that displayed a pattern of dominantly inherited ALS/motor neuron disease across four generations (4). Subsequent studies identified the mutation in at least seven different families, all of Portuguese-Brazilian origin, each displaying a different clinical course ranging from late-onset spinal muscular atrophy (SMA) to typical and atypical ALS (4). Our previous work identified only a single case of a VAPB mutation (P56S) in a screen of 80 familial ALS samples, demonstrating that VAPB mutations are extremely rare (5). As such, why is it important to study a mutation which is only responsible for a small percentage of ALS cases?
One reason is due to the fact that from a clinical point of view, familial and sporadic ALS cases are virtually identical. As such, it is not unreasonable to postulate that although ALS may be caused by different genetic factors, they all may lead to common sets of pathways that eventually result in the ALS phenotype. Thus, a high level of importance should be placed on understanding the common features of all known ALS genes since they may shed light on these pathways. Therefore, even though VAPB mutations are indeed rare, characterizing their effects may provide insight on how cases of ALS develop overall.
In both of the papers presented (6,7), the authors have each developed a Drosophila model of ALS which expresses mutant VAPB. The use of these models will undoubtedly be beneficial in future experiments to further decipher the ALS phenotype. Of great significance, though, is that each study observes in vivo that mutant VAPB is capable of inducing intracellular aggregates. This work reinforces previously published observation that in vitro expression of mutant human P56S protein results in cellular aggregates (4,5). The fact that the aggregation phenotype of this mutation is conserved down to Drosophila is quite interesting. Aggregates are commonly observed within ALS cases, as well as other neurodegenerative diseases, although whether these aggregates are pathogenic is still up for debate. The formation of intracellular aggregates has also been observed via expression of mutant SOD1 and mutant TDP-43 (3). Taken together, the observation that three different familial ALS genes all are capable of inducing intracellular aggregates reinforces the notion that understanding the activation of pathways by protein misfolding is key to understanding the pathogenic nature of ALS.
References:
1. Sreedharan J, Blair IP, Tripathi VB, Hu X, Vance C, Rogelj B, Ackerley S, Durnall JC, Williams KL, Buratti E, Baralle F, de Belleroche J, Mitchell JD, Leigh PN, Al-Chalabi A, Miller CC, Nicholson G, Shaw CE. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science. 2008 Mar 21;319(5870):1668-72. Abstract
2. Kabashi E, Valdmanis PN, Dion P, Spiegelman D, McConkey BJ, Vande Velde C, Bouchard JP, Lacomblez L, Pochigaeva K, Salachas F, Pradat PF, Camu W, Meininger V, Dupre N, Rouleau GA. TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet. 2008 May;40(5):572-4. Abstract
3. Winton MJ, Van Deerlin VM, Kwong LK, Yuan W, Wood EM, Yu CE, Schellenberg GD, Rademakers R, Caselli R, Karydas A, Trojanowski JQ, Miller BL, Lee VM. A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro. FEBS Lett. 2008 Jun 25;582(15):2252-6. Abstract
4. Nishimura AL, Mitne-Neto M, Silva HC, Richieri-Costa A, Middleton S, Cascio D, Kok F, Oliveira JR, Gillingwater T, Webb J, Skehel P, Zatz M. A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am J Hum Genet. 2004 Nov;75(5):822-31. Abstract
5. Landers JE, Leclerc AL, Shi L, Virkud A, Cho T, Maxwell MM, Henry AF, Polak M, Glass JD, Kwiatkowski TJ, Al-Chalabi A, Shaw CE, Leigh PN, Rodriguez-Leyza I, McKenna-Yasek D, Sapp PC, Brown RH Jr. New VAPB deletion variant and exclusion of VAPB mutations in familial ALS. Neurology. 2008 Apr 1;70(14):1179-85. Abstract
6. Tsuda H, Han SM, Yang Y, Tong C, Lin YQ, Mohan K, Haueter C, Zoghbi A, Harati Y, Kwan J, Miller MA, Bellen HJ. The amyotrophic lateral sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors. Cell. 2008 Jun 13;133(6):963-77. Abstract
7. Ratnaparkhi A, Lawless GM, Schweizer FE, Golshani P, Jackson GR. A Drosophila model of ALS: human ALS-associated mutation in VAP33A suggests a dominant negative mechanism. PLoS ONE. 2008 Jun 4;3(6):e2334. Abstract
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Related News: No Metal, No Stability: Structure of Apo SOD1
Comment by: Yoshiaki Furukawa
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Submitted 14 April 2009
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Posted 14 April 2009
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This study characterizes the dynamic behavior of SOD1 in detail. First, it essentially reproduces previous studies including the ones from the authors' group, as it has been well known that overall structures are similar between wild-type and mutant SOD1 proteins. In addition, significant differences in the dynamic behavior have been observed between Apo and holo forms of SOD1. When the metal ions are removed from the protein, structural disorder increases particularly in the loop regions.
We think that one of the interesting findings in this paper is the increased solvent accessibility of Cys-6 upon metal removal. Cys-6 is one of the four Cys residues (Cys-6, 57, 111, 146) in SOD1 and is buried toward the protein interior in the holo form of SOD1. In an enzymatically active form of SOD1, an intra-molecular disulfide forms between Cys-57 and 146, while Cys-6 and 111 remain reduced. In contrast, pathological inclusions purified from several ALS-model mice contain SOD1 multimers that are cross-linked via non-physiological disulfide bonds (
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This study characterizes the dynamic behavior of SOD1 in detail. First, it essentially reproduces previous studies including the ones from the authors' group, as it has been well known that overall structures are similar between wild-type and mutant SOD1 proteins. In addition, significant differences in the dynamic behavior have been observed between Apo and holo forms of SOD1. When the metal ions are removed from the protein, structural disorder increases particularly in the loop regions.
We think that one of the interesting findings in this paper is the increased solvent accessibility of Cys-6 upon metal removal. Cys-6 is one of the four Cys residues (Cys-6, 57, 111, 146) in SOD1 and is buried toward the protein interior in the holo form of SOD1. In an enzymatically active form of SOD1, an intra-molecular disulfide forms between Cys-57 and 146, while Cys-6 and 111 remain reduced. In contrast, pathological inclusions purified from several ALS-model mice contain SOD1 multimers that are cross-linked via non-physiological disulfide bonds (Furukawa et al., 2006).
It is, however, still controversial which Cys residues are involved in the formation of cross-linked SOD1 multimers under pathological conditions. While we have previously reported that the disulfide formation is not absolutely required for triggering SOD1 aggregation (Furukawa et al., 2008), an important role of Cys-6 and 111 in the formation of disulfide cross-links has been also suggested in the cultured cell model (Niwa et al., 2007). In addition, ALS-causing mutations at position 6 have been reported (i.e., C6G and C6F), implying that the other Cys residues are involved in the formation of disulfide-linked multimers even when Cys-6 is unavailable for disulfide formation. Nonetheless, the increased flexibility and solvent accessibility of Cys-6 upon metal removal will be an important clue to explain a molecular mechanism of the pathological SOD1 oligomer formation.
View all comments by Yoshiaki Furukawa |
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Related News: Chromogranin B: The ApoE of ALS?
Comment by: Pico Caroni
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Submitted 14 December 2009
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Posted 14 December 2009
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This important study establishes for the first time a genetic risk factor for sporadic ALS, thus providing a long-sought entry point into mechanisms and genetics of ALS. Genetic risk factors for neurodegenerative diseases will likely lead to novel insights into mechanisms of disease.
The impact of the chromogranin B mutations is slightly lower than that of the ApoE4 allele in Alzheimer disease. Because the chromogranin B mutations had an impact on onset time in familial ALS, and on risk of disease in sporadic ALS, the findings provide important support for the notion that sporadic and familial ALS are mechanistically related.
Chromogranin has been linked to mutant SOD1 by two previous studies. A frequently raised question is to what extent SOD1-based ALS mice are relevant in mimicking human disease. The association to chromogranin B in the SOD1 mouse model and now patient cohorts for both sporadic and familial ALS strongly supports the notion of converging cellular and molecular mechanisms of disease.
One important implication for scientists working on ALS is that the...
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This important study establishes for the first time a genetic risk factor for sporadic ALS, thus providing a long-sought entry point into mechanisms and genetics of ALS. Genetic risk factors for neurodegenerative diseases will likely lead to novel insights into mechanisms of disease.
The impact of the chromogranin B mutations is slightly lower than that of the ApoE4 allele in Alzheimer disease. Because the chromogranin B mutations had an impact on onset time in familial ALS, and on risk of disease in sporadic ALS, the findings provide important support for the notion that sporadic and familial ALS are mechanistically related.
Chromogranin has been linked to mutant SOD1 by two previous studies. A frequently raised question is to what extent SOD1-based ALS mice are relevant in mimicking human disease. The association to chromogranin B in the SOD1 mouse model and now patient cohorts for both sporadic and familial ALS strongly supports the notion of converging cellular and molecular mechanisms of disease.
One important implication for scientists working on ALS is that the research mechanistically links SOD1 mutations to a set of mutations in chromogranin B. We now have potentially interacting mutations in two genes to study; this will likely lead to new disease models and hopefully to first elements of a molecular disease pathway. It will be interesting to determine whether there is a relationship between mutant SOD1 secretion and chromogranin B mutations. Should this be the case, it may suggest that the risk involves local secretion of misfolded proteins such as mutant SOD1. Of further interest are the implications for the role of ER stress pathways in the pathogenesis of ALS.
It will be important to determine whether other genes implicated in ALS (including the RNA metabolism genes TDP-43 and FUS) also synergize with chromogranin B mutations to promote disease. Whether chromogranin A variants link to ALS is definitely worth investigating as well.
View all comments by Pico Caroni |
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Related News: News Brief: Biomedical Funding Dips, AD Research Feels the Pinch
Comment by: Meghan Kallman
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Submitted 21 January 2010
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Posted 21 January 2010
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In addition to the traditional funding models mentioned in this story, we would like to also mention a new funding model available for ALS research. Prize4Life is a nonprofit organization that awards two prizes of $1 million each (the ALS Biomarker Challenge and the ALS Treatment Prize). Instead of recognizing historical accomplishments, Prize4Life designs prizes that we believe are achievable in a two- to three-year timeframe and then recruits teams to compete. Prize competitions have been steadily gaining traction in a variety of domains of innovation because their emphasis on specific outcomes has the capacity to propel a field forward very quickly, and can attract creative thinking from both within a field and “outside the box.” For example, in 2009 Prize4Life awarded two $50,000 Milestone Prizes, one of which went to an established ALS researcher, and one of which went to a trained dermatologist who explored a completely novel approach towards an ALS biomarker. Visit Prize4Life to learn more or to register to compete for a...
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In addition to the traditional funding models mentioned in this story, we would like to also mention a new funding model available for ALS research. Prize4Life is a nonprofit organization that awards two prizes of $1 million each (the ALS Biomarker Challenge and the ALS Treatment Prize). Instead of recognizing historical accomplishments, Prize4Life designs prizes that we believe are achievable in a two- to three-year timeframe and then recruits teams to compete. Prize competitions have been steadily gaining traction in a variety of domains of innovation because their emphasis on specific outcomes has the capacity to propel a field forward very quickly, and can attract creative thinking from both within a field and “outside the box.” For example, in 2009 Prize4Life awarded two $50,000 Milestone Prizes, one of which went to an established ALS researcher, and one of which went to a trained dermatologist who explored a completely novel approach towards an ALS biomarker. Visit Prize4Life to learn more or to register to compete for a prize. View all comments by Meghan Kallman |
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Related News: Glutamate Gums Up Motor, Dopaminergic Neurons
Comment by: Walter Bradley DM, FRCP
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Submitted 29 April 2010
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Posted 29 April 2010
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I recommend the Primary Papers
Amber Dance has nicely summarized both papers. Concerning the Cucchiaroni paper, I believe it is a very important contribution to the literature. Deborah Mash, John Pablo, and colleagues have recently confirmed prior studies by Paul Cox and colleagues that BMAA is accumulated in the brains of patients with Alzheimer disease. They also showed that BMAA is accumulated in the brains of patients with amyotrophic lateral sclerosis, but not in brains of control patients or those with Huntington disease. They have obtained preliminary evidence that BMAA is accumulated in the brains of patients with Parkinson disease.
The concentration of BMAA in the protein-bound fraction of the brains of Chamorros with ALS/PDC is of the order of 5 mM, while that in the brains of patients with AD, PD, and ALS is approaching 1 mM. However, Cox and colleagues found that the concentration of BMAA in the soluble fraction of the brains of Chamorros with ALS/PDC was about 50 micromolar.
The paper by Cucchiaroni et al. demonstrates a significant physiological effect of BMAA on dopaminergic neurons. It...
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Amber Dance has nicely summarized both papers. Concerning the Cucchiaroni paper, I believe it is a very important contribution to the literature. Deborah Mash, John Pablo, and colleagues have recently confirmed prior studies by Paul Cox and colleagues that BMAA is accumulated in the brains of patients with Alzheimer disease. They also showed that BMAA is accumulated in the brains of patients with amyotrophic lateral sclerosis, but not in brains of control patients or those with Huntington disease. They have obtained preliminary evidence that BMAA is accumulated in the brains of patients with Parkinson disease.
The concentration of BMAA in the protein-bound fraction of the brains of Chamorros with ALS/PDC is of the order of 5 mM, while that in the brains of patients with AD, PD, and ALS is approaching 1 mM. However, Cox and colleagues found that the concentration of BMAA in the soluble fraction of the brains of Chamorros with ALS/PDC was about 50 micromolar.
The paper by Cucchiaroni et al. demonstrates a significant physiological effect of BMAA on dopaminergic neurons. It suggests that a major source of this effect is via the GluR1 subunit and provides a vital link in the pathway from the environmental neurotoxin—BMAA derived from ubiquitous cyanobacteria—to the human diseases AD, PD, and ALS. However, it should be noted that the concentrations used in the study were in the mM range; more chronic, lower-dose studies are needed to simulate the apparent state in humans.
The field of cyanobacterial BMAA and its role in human neurodegenerative diseases was recently reviewed in a supplement to the journal ALS.
References:
Pablo J, Banack SA, Cox PA, Johnson TE, Papapetropoulos S, Bradley WG, Buck A, Mash DC. Cyanobacterial neurotoxin BMAA in ALS and Alzheimer’s disease. Acta Neurol Scand 2009;120:216-225. Abstract
Murch SJ, Cox PA, Banack SA, Steele JC, Sacks OW. Occurrence of beta-methylamino-l-alanine (BMAA) in ALS/PDC patients from Guam. Acta Neurol Scand 2004a;110 (4):267–269. Abstract
Murch, S.J., Cox, P.A., Banack, S.A. A mechanism for slow release of biomagnified cyanobacterial neurotoxins and neurodegenerative disease in Guam. Proceedings of the National Academy of Sciences 2004b; 101:12228-31. Abstract
Bradley, W.G., Cox, P.A. Supplement 2. Amyotrophic Lateral Sclerosis. June 2009;10:1-128.
View all comments by Walter Bradley DM, FRCP |
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Related News: Another Screen, Another Gene: ALS and the Right-handed Serine
Comment by: Steve Barger
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Submitted 27 April 2010
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Posted 29 April 2010
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Regarding the statement in this news story "De Belleroche also pointed out that a DAO mutant mouse might be a useful model for ALS research, which sorely needs new models," I'd like to note that it is unlikely that loss of DAO alone will be sufficient for disease. A line of DAO mutant mice, essentially devoid of DAO activity, has been around almost 30 years (Konno and Yakumura, 1983) without any ALS-like symptoms reported in the first year of life. Here is a quote from that paper:
"No apparent difference was detected between DAO+ and DAO- mice. The DAO- mice grew and behaved normally. They were fertile and produced as many offspring as the DAO+ animals did. Besides, the unilaterally nephrectomized DAO- mice lived more than 1 year without any impairment of health. ...[T]he discovery of the DAO- mice suggests that the enzyme is not essential, at least for the growth and reproduction of the mouse under laboratory conditions."
References:
Konno R, Yasumura Y. 1983. Mouse mutant deficient in D-amino acid oxidase activity. Genetics 103:277-85. Abstract
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