London, Ontario: The Fuss About FUS at ALS Meeting

06 Jul 2009

As neurologists and neurobiologists gathered for the Third International Research Workshop on Frontotemporal Dementia in ALS, 21-25 June in London, Ontario, Canada, the big buzz was all about Fused in Sarcoma (FUS), the gene recently discovered to be mutated in 3 to 5 percent of people with inherited amyotrophic lateral sclerosis. Christopher Shaw of King’s College London described how, after a 10-year effort in collaboration with Robert Brown of the University of Massachusetts in Worcester, researchers plucked the FUS gene from a set of families with inherited ALS (see ARF related news story; Kwiatkowski et al., 2009 and Vance et al., 2009). The primarily nuclear protein is involved in transcribing, splicing, and transporting RNA; it is not yet clear what links these functions to motor neuron disease. Scientists have found more than a dozen mutations, mostly in the carboxyl-terminal region of the protein, which cause it to form inclusions in cultured cells as well as in human spinal cord. It has been just four months since that work was published, but others have already tackled FUS in their own studies.

Some people with ALS also get FTD, and the two conditions are linked by the pathologic protein TDP-43. Ian Mackenzie of the University of British Columbia in Vancouver presented new data on FUS pathology in frontotemporal dementia. FTD cases fall into two main categories: those with tau pathology, and those with TDP-43 pathology. But a small subset of cases fit neither category, with ubiquitin-positive inclusions that lack both tau and TDP-43 (Mackenzie et al., 2008 and Roeber et al., 2008).

This atypical FTD has disease onset between 28 and 55 years of age and a quickly progressing condition that lasts between four and 15 years. The disease includes severe personality and behavioral changes, sometimes leading to antisocial or even criminal activity. It does not appear to be inherited.

Mackenzie and colleagues analyzed brain tissue from 15 people who had this atypical FTD and discovered “very unusual neuronal inclusions,” he said. In addition to round or crescent-shaped cytoplasmic inclusions, the scientists observed twisted, thick filaments inside the nucleus.

When they immunostained their atypical FTD samples for FUS, the normal pattern—strong signal in the nucleus, and some in the cytoplasm—was still apparent. However, there was additional staining for both the cytoplasmic and intranuclear inclusions. Inclusions did not stain positive for FUS in samples from people with FTD associated with tau or TDP-43. The scientists found no mutations in the FUS sequence of several people with atypical FTD. Mackenzie and colleagues discovered similar FUS pathology in samples from people with basophilic inclusion body disease and neuronal intermediate filament inclusion disease, which also cause frontotemporal dementia. These FUSopathies form a new molecular class of FTD, Mackenzie said, although it is not yet clear whether all are the same disease, or different conditions with similar pathology.

Some of the answers about FUS are likely to come from animal models, and at least a few are already in the works. Don Cleveland of the University of California, San Diego, reported on mouse models he is developing in collaboration with Shaw. He has mice expressing wild-type as well as mutant human FUS at varying levels, under control of the native or prion promoter, which drives expression in the nervous system. He noted that in animals that express high levels of the human transgene, it appears to dampen expression of mouse FUS, with less mouse protein present. In the nervous system, the protein is primarily nuclear, with a bit of cytoplasmic expression. In the case of mutant FUS, some protein formed aggregates in the spinal cord. Now, Cleveland said, it’s a waiting game. Some lines are only three months old, and he and his colleagues must be patient as they hope for a phenotype.—Amber Dance.

Comments

No Available Comments

Make a Comment

To make a comment you must login or register.

References

News Citations

Paper Citations

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. 2009 Feb 27;323(5918):1205-8. PubMed.
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. PubMed.
Mackenzie IR, Foti D, Woulfe J, Hurwitz TA. Atypical frontotemporal lobar degeneration with ubiquitin-positive, TDP-43-negative neuronal inclusions. Brain. 2008 May;131(Pt 5):1282-93. PubMed.
Roeber S, Mackenzie IR, Kretzschmar HA, Neumann M. TDP-43-negative FTLD-U is a significant new clinico-pathological subtype of FTLD. Acta Neuropathol. 2008 Aug;116(2):147-57. PubMed.

London, Ontario: TDP-43 Across the Animal Kingdom at ALS Meeting

06 Jul 2009

Since it was linked to amyotrophic lateral sclerosis in 2006, TAR DNA Binding Protein-43 has become a top player in research on ALS and frontotemporal dementia. People with both conditions often accumulate TDP-43 aggregates in neurons. These two TDP-43 proteinopathies, which some have suggested are simply the two ends of a continuous spectrum, were discussed in the Third International Research Workshop on Frontotemporal Dementia in ALS, held 21-25 June in London, Ontario. In order to figure out how TDP-43 causes disease, scientists need animal models, and there was no shortage of these in the presentations. “There’s probably never been a more exciting time to work on ALS,” said Don Cleveland of the University of California, San Diego.

Cleveland, as well as many other scientists, is working to develop a mouse model for TDP-43 disease. TDP-43 is an essential protein, so simple deletions are out. Cleveland presented his lab’s progress, in collaboration with Christopher Shaw of King’s College London, on a set of transgenic mice that carry human TDP-43 along with the endogenous mouse gene. The animals express the transgene, in varying amounts, but there have been a few snags in establishing lines. Several founders failed to produce progeny. In animals expressing TDP-43 containing mutations found in human disease, the researchers have been able to make fertile animals with symptoms of neurodegenerative disease, but the phenotype is weaker in the F1 and F2 generations.

While the field anxiously awaits a useful mouse model, plenty of other animals are ready for experimentation now. Ronald Klein of the Louisiana State University Health Sciences Center in Shreveport presented his rat model, in which he uses a adeno-associated virus to transfer the human TDP-43 gene into the substantia nigra (see Tatom et al., 2009 and ARF related news story). Klein hopes to improve his model by adding a “trigger” factor that will cause the human TDP-43 to more readily leave the nucleus for the cytoplasm, as it does in disease.

Cleveland has begun using a somewhat similar approach in rodents, except his goal is to silence TDP-43 expression, to see what consequences this has for RNA regulation and physiology. By pumping antisense oligonucleotides into the cerebral spinal fluid, scientists in the Cleveland lab were able to decrease TDP-43 expression. With TDP-43 RNA interference, the initial experiments proved the protein was essential: as the levels dropped, the animals eventually died. “This was actually a disaster,” Cleveland said. “We didn’t want them to be dead.”

Another option used by Shaw is chick embryo (see Sreedharan et al., 2008 and ARF related news story). The model relies on electroporation to transfect tagged transgenes into the spinal cord. Conveniently, the genes can be targeted to only one side of the spinal cord, allowing the other side to serve as an internal control. Shaw reported that his group has overexpressed mutant TDP-43 in more than 300 embryos, with a consistent result—cell death. Using GFP-tagged versions of TDP-43, Shaw’s lab found that the wild-type protein remained in the nucleus where it belonged, while mutants were found mainly in the cytoplasm. Some mutant TDP-43 remained in the nucleus, where it formed inclusions.

Zebrafish can also provide useful information, said Philip Van Damme of VIB in Leuven, Belgium. He uses zebrafish embryos “mainly because it’s so easy,” he said—the animals are small and cheap, and convenient tools are available. Using oligonucleotides called morpholinos, scientists can block translation of mRNA. Alternatively, they can inject RNA to ramp up gene expression. There are two homologs of TDP-43 in zebrafish: TARDBP shares 74 percent homology, and TARDBPL 57 percent, with the human gene. Overexpression of wild-type human TDP-43 resulted in shorter motor neuron axons with aberrant branching, and mutant versions of TDP-43 exacerbated the phenotype. These problems matched those of animals overexpressing SOD1, another gene linked to ALS (see Lemmens et al., 2007). “We were really pleased to see we could obtain a phenotype similar to the one we saw before with SOD1,” Van Damme said. He suggested the zebrafish would make a useful “pre-model”—allowing scientists to test their hypotheses quickly before moving into time-consuming mouse studies.

There is plenty to be learned from invertebrates as well. David Morton of the Oregon Health & Science University in Portland and Emanuele Buratti of the International Centre for Genetic Engineering and Biotechnology (ICGEB) in Trieste, Italy, both presented results from studies in Drosophila. “What we’re trying to do is basically use Drosophila to understand the normal functions of TDP-43,” Morton said. “Ultimately, we would really like to understand what is going on at a cellular level.” Fruit flies also boast a host of useful genetic and genomic tools. By mixing and matching ready-made promoter-gene fusions, insect biologists can express their target gene across a variety of tissues, or knock it out via RNA interference if they prefer.

The Drosophila version of TDP-43 is TBPH. Among the 46 percent of residues conserved between human and fly are 11 of the 28 residues shown to be mutated in some forms of human disease. Silencing TBPH across all tissue types with RNA interference killed flies before adulthood, but knocking down TBPH expression selectively in neurons, by a factor of two or three, resulted in viable animals, Morton found. The flies showed neurodegeneration in the retina and lesions in the neuropil that increased with age. Although Morton’s TBPH deletions, like the RNAi knockdown models, died before reaching adulthood, the scientists were able to study them as larvae. “We saw a dramatic reduction of the total distance crawled by these larvae,” Morton reported, although just by watching the animals their process of movement appears to be normal.

In contrast, Buratti had data on TBPH knockouts that did survive to adulthood, from the laboratory of Fabian Feiguin at ICGEB. “It’s something that [we] have to sort out,” Morton said. He suspects that there may only be minor differences between the lines, though, because his knockouts developed into fully formed adults that apparently lacked the strength to climb out of the pupal cuticle after metamorphosis. Feiguin’s flies were also weak, often needing assistance in exiting the pupal cuticle, and had reduced lifespans. In Feiguin’s experiments, adults that had reduced expression of TBPH in neurons due to RNA interference also had motility defects. Co-expression of TBPH, or human TDP-43, somewhat rescued these defects.

Worms, too, have something to offer in the TDP-43 arena. Human TDP-43 causes movement defects when expressed in the nematode C. elegans, reported Brian Kraemer of the University of Washington in Seattle. They wriggled only half as far across a plate as non-transgenic animals. When the TDP-43 transgene contained disease-associated mutations, the phenotype intensified, with some animals barely traversing a millimeter. “They don’t get around very well at all,” Kraemer said. Worms also have their own TDP-43, but it lacks the carboxyl-terminal region where the disease-causing mutations cluster. The transgenic worms show several features of human disease, Kraemer said, including progressive motor neuron dysfunction and degeneration, decreased lifespan, and accumulation of aggregated, insoluble TDP-43. Worm models can be used to understand the basic biology of TDP-43 pathology in a simple system with only 302 neurons, he said, as well as to search for genes that interact with the TDP-43 pathway.

Each of these systems offers a platform for asking the most basic questions about TDP-43: What happens when its function is lost in the nucleus? How does it cause pathology? How do mutations contribute to these events? And ultimately, how can medicine halt these molecular disasters? Having fashioned the tools, scientists can now apply them to screen therapies as well as investigate basic biology.—Amber Dance.

Comments

No Available Comments

Make a Comment

To make a comment you must login or register.

References

News Citations

Paper Citations

Tatom JB, Wang DB, Dayton RD, Skalli O, Hutton ML, Dickson DW, Klein RL. Mimicking aspects of frontotemporal lobar degeneration and Lou Gehrig's disease in rats via TDP-43 overexpression. Mol Ther. 2009 Apr;17(4):607-13. PubMed.
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. Epub 2008 Feb 28 PubMed.
Lemmens R, Van Hoecke A, Hersmus N, Geelen V, D'Hollander I, Thijs V, Van Den Bosch L, Carmeliet P, Robberecht W. Overexpression of mutant superoxide dismutase 1 causes a motor axonopathy in the zebrafish. Hum Mol Genet. 2007 Oct 1;16(19):2359-65. PubMed.

London, Ontario: More Than Motor Malaise at ALS Meeting

10 Jul 2009

Say “amyotrophic lateral sclerosis” and many doctors will think of the motor impairments that ultimately cause paralysis. But in a significant subset of cases, the mind is also affected. Some people with ALS struggle with cognitive tasks, such making word lists. A growing understanding of cognitive impairment in ALS made a strong showing at the Third International Research Workshop on Frontotemporal Dementia in ALS, held in London, Ontario, from 21-25 June.

If only a subset of patients will show cognitive symptoms, who will they be? Paul Schulz of the Baylor College of Medicine in Houston, Texas, presented several clues in his presentation on risk factors for cognitive impairment in ALS. Schulz and collaborators have found that cognitive symptoms correlated with ApoE genotype and diabetes.

A decade ago, few neurologists considered the cognitive side of ALS, and that attitude persists in many medical school classrooms and patient resources, Schulz said. When a colleague, Paul Massman of the University of Texas at Austin,published evidence of neuropsychological deficits in people with ALS (Massman et al., 1996), Schulz was rather embarrassed he had not noticed the symptoms in his patients; most doctors assumed their slower responses were due to the trouble they had moving or speaking.

The exact rates of cognitive impairment in ALS are uncertain because methods for assessing cognition vary, said Orla Hardiman of Trinity College in Dublin, Ireland, who did not attend the meeting. In addition, most studies have been done at centers focused on cognition, which attract more patients with such impairment, potentially skewing the results. Her (unpublished) and Schulz’s (Ringholz et al., 2005) work indicate that one-third to one-half of people with ALS are cognitively normal. The rest have some signs of cognitive or behavioral involvement, and perhaps 10-20 percent meet the criteria for frontotemporal dementia.

Understanding cognitive symptoms in ALS is important to both patients and scientists, Hardiman said. People with ALS and their families may want to make decisions about end-stage care early, before mental impairment arises. And understanding the different forms of the disease is essential for clinical trials, Hardiman said: “If we don’t stratify for cognitive impairment in clinical trials, we are actually probably introducing confounders into the study.”

The full neuropsychological battery to define cognitive skills can cost $2,000-$4,000, Schulz said, so it is worth knowing who is at risk before committing to testing. Some scientists at the meeting presented short questionnaires to screen people with ALS for cognitive symptoms. Schulz is trying to understand the risk factors that might point to a heightened chance of mental involvement. Then, basic scientists can work backwards from his clinical results to figure out how those factors influence disease.

One of the first factors Schulz tackled was a genetic basis for ALS. Approximately one-tenth of patients have a family history of disease. Schulz surmised that the familial cases would be similar to each other with respect to cognitive symptoms: “I figured it would be either everybody or nobody.” But instead, he and his collaborators found the rates of cognitive dysfunction among 37 people with familial ALS to be about the same as 392 spontaneous cases, with two-thirds of familial cases and one half of spontaneous cases exhibiting cognitive impairment(see Wheaton et al., 2007). “To be honest, I was very surprised.” It is still possible that among the familial cases, mutations in certain genes have specific effects on cognition; the researchers are currently genotyping subjects.

Schulz hypothesized that ApoE genotype, which can make a 20-year difference to onset of Alzheimer disease, might be involved in ALS as well. When he and his colleagues compared ApoE genotypes to age of onset and speed of progression of motor symptoms in 852 people with spontaneous ALS, they found no effect. “There was not a single shard of difference between the various ApoE genotypes,” Schulz said. But cognitive testing of 185 subjects in the same group gave them more exciting results: people with at least one ApoE4 allele performed worse on six different tasks. So ApoE4 may be a risk factor for cognitive impairment in ALS, although, Schulz said, the association is “not strong enough to bet the farm on.”

Schulz suspected metabolism, too, might play a role, since diabetes is a risk factor for dementia (see ARF related news story) and hypermetabolism has been linked to ALS ARF related news story). In a study of 175 diabetics and 2,196 non-diabetics, he found another surprise. Diabetics were generally diagnosed with ALS at a slightly older age than those without diabetes. “That’s the shocker here—four years later age of onset,” he said. But the subset that underwent cognitive testing—24 people with ALS and diabetes, 429 with ALS only—showed a different story. Diabetics performed worse on cognitive tests at the time of diagnosis with ALS. “There’s something funny going on in diabetes,” Schulz said, although he is not sure how to explain the results. Perhaps, he guessed, the altered blood sugar levels in diabetics protects motor neurons, but makes other neurons more vulnerable.

Metabolism and weight control are often problematic in people with ALS. Hypermetabolism can cause patients to burn too many calories, and the disease often interferes with swallowing, so patients frequently lose weight. But Schulz and colleagues have found that those who maintained their body mass index (BMI) had increased survival of up to nine months. Of course, the correlation could go in either direction: people who keep their weight up could be protected, or people with a slower disease might find it easier to maintain weight. Diabetes, Schulz surmised, might counter the effects of hypermetabolism, and thus be protective. He has not yet looked for a link between body mass and cognitive function, but hopes to test a weight-maintenance program to see if it slows disease in people with ALS.

One risk factor will probably not be enough to predict cognitive impairment in ALS patients, but a collection of factors could be significant, Schulz said. He is also looking into social cognition tasks, ethnicity, traumatic brain injury and other neurodegenerative conditions as possible tip-offs to look for cognitive impairment. “This research helps clinicians determine which ALS patients may be at greater risk for cognitive impairment, so they can better triage patients for neuropsychological testing or counseling,” wrote Susan Woolley of the Forbes Norris ALS Research Center in San Francisco, in an e-mail to ARF. Woolley attended the meeting but is not involved in Schulz’s studies.

Schulz also hopes his work will inspire basic scientists studying ALS. ApoE genotype and metabolic effects should have a place at the lab bench, he said; for example, it would be interesting to cross an ALS mouse model with a diabetes model and look for changes in motor and cognitive symptoms. Denise Figlewicz, vice president for research at the ALS Society of Canada (one of the sponsors of the June meeting), agrees. “The question of calorie consumption and overall metabolism with respect to ALS patients has been a very dynamic area in the past few years,” she wrote in an e-mail to ARF. “In addition to ‘pushing’ patients to be sure to take in enough calories, I hope that more people will get involved in studying what the data we have so far really means.”

Doctors and scientists are beginning to realize that cognition is an important part of ALS. “We are starting to go beyond the phase of wondering if there is cognitive impairment,” Schulz said. “Now we are going on to the next step, to figure out who is at risk for it, and why they are at risk.”—Amber Dance.

Comments

No Available Comments

Make a Comment

To make a comment you must login or register.

References

News Citations

Paper Citations

Massman PJ, Sims J, Cooke N, Haverkamp LJ, Appel V, Appel SH. Prevalence and correlates of neuropsychological deficits in amyotrophic lateral sclerosis. J Neurol Neurosurg Psychiatry. 1996 Nov;61(5):450-5. PubMed.
Ringholz GM, Appel SH, Bradshaw M, Cooke NA, Mosnik DM, Schulz PE. Prevalence and patterns of cognitive impairment in sporadic ALS. Neurology. 2005 Aug 23;65(4):586-90. PubMed.
Wheaton MW, Salamone AR, Mosnik DM, McDonald RO, Appel SH, Schmolck HI, Ringholz GM, Schulz PE. Cognitive impairment in familial ALS. Neurology. 2007 Oct 2;69(14):1411-7. PubMed.

International Research Workshop on Frontotemporal Dementia in ALS

Tools

London, Ontario: The Fuss About FUS at ALS Meeting

Comments

Make a Comment

References

News Citations

Paper Citations

Further Reading

Papers

News

London, Ontario: TDP-43 Across the Animal Kingdom at ALS Meeting

Comments

Make a Comment

References

News Citations

Paper Citations

Further Reading

Papers

News

London, Ontario: More Than Motor Malaise at ALS Meeting

Comments

Make a Comment

References

News Citations

Paper Citations

Further Reading

Papers

News