Are TDP-43 Mice Living Up to Expectations?

20 Sep 2012

Mouse modelers wasted no time when, five years ago, the TAR DNA binding protein 43 proved not only to be mutated in some cases of amyotrophic lateral sclerosis (ALS), but also to be deposited broadly in inclusions in the majority of sporadic ALS, and even a subset of frontotemporal dementia (FTD). Researchers immediately hoped this discovery would lead to a powerful new disease model that could overcome the flaws of the tired workhorse of ALS preclinical studies. For more than a decade, ALS researchers had been limited to mice expressing mutant human superoxide dismutase 1 (SOD1), which represent but a small fraction of familial ALS cases. Worse, experiments in the SOD1 mice had failed to yield desperately needed medicines to slow the disease (see ARF Webinar). Many labs raced to engineer new mouse lines.

“We jumped into the TDP-43 modeling scenario … very quickly after the gene was discovered,” noted Jada Lewis, of the University of Florida in Gainesville, in a presentation at the 8th International Conference on Frontotemporal Dementias held 5-7 September 2012 in Manchester, U.K. But then—and now—researchers did not fully understand how TDP-43 contributes to ALS and FTD. Half a decade later, how are the new lines measuring up? Not quite as high as some had hoped. Scientists who are partway through characterizing the earliest models have discovered unexpected phenotypes that could nix their dream to test drugs in certain TDP-43 mice. But to be fair, Lewis noted that five years is a short time in the world of mouse modeling. In comparison, it took 20 years for scientists to come up with a good model for the hemoglobin mutations responsible for sickle cell anemia, she said. The take-home message for scientists is that rigorous characterization of a mouse model should precede its use in drug discovery.

With TDP-43, Robert Baloh of Cedars-Sinai Medical Center in Los Angeles was first out of the gates. His model expresses an alanine-315-threonine substitution also found in human ALS. Researchers greeted the publication with considerable excitement (see ARF related news story on Wegorzewska et al., 2009). “There were high expectations. People were thinking these mice would start getting flagrant FTD and ALS,” Baloh said. “The problem is, they are still mice. Humans and mice, even if they have the same genetic mutation, get different diseases.” In further analyses, researchers are finding that it is not neurodegeneration that kills Baloh’s mice. Instead, they die from bowel obstruction.

Disheartening revelations such as this are coming out as researchers breed the animals with standard strains and drill deeper into their pathology and symptoms than the paper initially reporting the new model was able to do. The Jackson Laboratory in Bar Harbor, Maine (JAX), breeds and distributes Baloh’s mice; they are also analyzing the animals’ phenotype.

The initial publication on a new model is just a description. At that stage, the mouse is not ready for drug studies, said Steven Perrin of the ALS Therapy Development Institute in Cambridge, Massachusetts. Like JAX, ALS TDI is breeding and characterizing the Baloh model. They are also doing that for another early model of the TDP-43 rush, from Jeffrey Elliott’s lab at the University of Texas Southwestern Medical Center in Dallas. Elliott’s group produced A315T, methionine-337-valine, and wild-type versions of TDP-43 mice; ALS TDI has started working with the wild-type so far (Stallings et al., 2010). ALS TDI expects to issue recommendations on how researchers should conduct preclinical studies with TDP-43 mice.

To date, at least 10 TDP-43 lines have been reported in the literature (reviewed in Cohen et al., 2011 and Wegorzewska and Baloh, 2011). As a group, they were difficult to obtain because the protein’s role in development makes knockouts difficult, and their ability to regulate their own protein level complicates overexpression. Broadly speaking, the lines share many features. Most seem akin to ALS, although researchers can push them toward an FTD phenotype by using a forebrain-specific promoter. The animals tend to develop trouble moving and die young. They suffer neurodegeneration and neuroinflammation. Ubiquitinated inclusions usually speckle their neurons. TDP-43 protein vacates the nucleus and fragments, though oddly, it does not join ubiquitin in those aggregates as frequently as it does in human tissue samples.

“The mice reproduce a great deal of the pathology … and a large part of the disease phenotype,” Elliott said. He thinks TDP-43 transgenic mice more closely resemble sporadic ALS than does the SOD1 model, because the mice get sick regardless of whether they express mutant or wild-type TDP-43. “That is an important feature,” Elliott said, since the majority of people with ALS have wild-type TDP-43. In contrast, mice with wild-type human SOD1 develop only minimal disease and live a full lifespan.

Quality Control
But reproducing pathology is only the first step. Subsequent treatment studies with a given mouse model also need to meet certain quality standards. For example, in the first decade of SOD1 mouse research, scientists across the field tested treatments with as few as four mice per treatment arm, Perrin told Alzforum. These studies generated papers in top-tier journals but did not reproduce in larger validation studies, much less in clinical trials. It was not until 2008, when ALS TDI published a study based on 5,429 mice from the SOD1-glycine-93-alanine strain, that researchers learned it would take at least 24 animals per group to robustly measure a 5 percent improvement in survival (Scott et al., 2008; see also ARF Webinar). Those groups should be balanced for males and females, because males sicken more quickly. Perrin further recommends leaving out animals that die early of something other than neurodegeneration, as that confounds results.

ALS TDI’s paper was “a big eye opener,” said Cat Lutz of JAX. Since 2008, awareness of a problem with reproducibility of mouse treatment studies has spread to the Alzheimer’s research community and beyond (see, e.g. (Shineman et al., 2011). Modern ALS studies tend to follow ALS TDI’s guidelines, but it is too soon to tell if the improvements in preclinical research herald success for clinical trials, Perrin said.

In applying the same rigor of characterization to TDP-43 mice, scientists at JAX and ALS TDI first had to stabilize the genetic background of Baloh’s animals. Scientists frequently generate new mutants in hybrid strains because these are convenient to work with, Lutz said. For example, their embryos have large nuclei that are easy to inject. But genetic background can greatly affect phenotype, so the ALS TDI and JAX teams bred the new model with standard black 6 (C57BL/6J) mice for several generations. Making Baloh’s mice congenic has made them better candidates for drug studies, Lutz said. Originally, their lifespan varied greatly, but moving the mutation into the black 6 background made it tighter and reproducible.

The first challenge in readying the line for drug research turned out to be simply breeding a sufficient number of mice. “That took a lot longer than we had anticipated,” said Fernando Vieira of ALS TDI. Typically, 80 percent of male-female pairs will yield litters, but for the TDP-43 mice, only 40 percent did. Part of the problem is that the transgenic males fell ill so quickly, they were not around very long to breed. At this point in ALS TDI’s studies, males appear to live for about 100 days; females typically die at 130 days but some make it out to 210, Vieira said. Wild-type lab mice live at least two years.

The researchers are unsure why breeding was so difficult. Vieira noted that the prion promoter controlling the transgene would turn on mutant TDP-43 in the testes; many males suffered odd abscesses near their genitals that may have killed the mood, so to speak. After struggling to produce approximately 300 mice the natural way, ALS TDI has resorted to in-vitro fertilization, and is in the process of obtaining 600 more animals, Vieira said.

Gut, Not Brain, Dooms ALS Mouse
To their surprise, the ALS TDI and JAX teams observed that the Baloh mice do not die of progressive neurodegeneration. Technicians were puzzled when they noticed animals went from health to death within a couple of days. The cause turned out to be bowel obstruction. “They feel lumpy,” Vieira said. “Their guts are completely filled with food.” Others have observed this as well (Guo et al., 2012).

The symptom is intestinal, but the root cause is likely neuronal. At JAX, researchers observed that the gut of the model mice might contain fewer autonomic ganglion cells, though Lutz cautioned that the finding is preliminary. This would suggest that the autonomic nervous system, which controls how the intestine moves food, stalls in the TDP-43 mice. “There is nothing wrong with the gut itself,” Baloh said. “It is a control problem of the gut.” This is not known to be a symptom of human ALS, although occasionally people do report digestive problems.

ALS TDI has not been studying Elliott's wild-type TDP-43 model as long as Baloh’s. Elliott’s also uses the prion promoter, but thus far the researchers have not seen bowel obstruction. “Why is the Elliott mouse different from the Baloh mouse?” Perrin asked. After all, the transgenic approach behind both is similar. He thinks it might have to do with the level of transgene expression—it is higher in Baloh’s strain—or with the locus where human TDP-43 settled into the mouse chromosome.

Lewis was not surprised to hear of the bowel phenotype. She noticed the same problem with mice expressing high concentrations of transgenic tau, a line that also relied on the prion promoter. Lewis suspects that something about neurodegenerative proteins under the control of this promoter attacks the neurons that regulate the gut.

Degeneration Versus Development
Because of the bowel blockage, Baloh’s mice never reach the point where they would suffer the full-blown muscle atrophy and neurodegeneration typical of ALS, Vieira said. In the spinal cord, it appears there are fewer motor neurons than normal, but those that are present show no signs of degeneration or apoptosis. He speculated the missing motor neurons might never have developed in the first place. “There is something going on there, but I do not know if it is actually motor neuron degeneration,” he said.

Like Baloh’s, most models are based on a TDP-43 transgene that is active from birth. These always-on transgenics can certainly tell scientists about TDP-43’s physiological role, Lewis said. “But for disease relevance, we need to get past what TDP-43 does in development.” Like so much about the protein’s function, it is not certain what it is doing in development, but the gene clearly is important since knockout embryos do not survive.

At the Alzheimer’s Association International Conference, held 14-19 July 2012 in Vancouver, Canada, Lewis presented an alternate transgenic approach from her paper in Acta Neuropathologica in June (Cannon et al., 2012). The team created a repressible TDP-43 model with the protein expressed in the forebrain, to mimic FTD, unless the animals are fed doxycycline.

Mice on a doxy-free diet looked much like other TDP-43 models. They had smaller brains, though the tissue did not shrink with age as would be expected for neurodegeneration. Instead, the growth rate of the transgenic mouse brains dropped between 12 and 24 days of age, when the brain is still developing. Other TDP-43 models also show low brain weight early in life (see ARF related news story on Xu et al., 2010; Xu et al., 2011).

When the team fed doxycycline to keep TDP-43 off until the mice were weaned at three weeks of age, they saw something quite different. The animals showed no physical symptoms, but in the brain, they progressively lost neurons, Lewis told Alzforum. Their brains atrophied to below normal weight by five and a half months of age, and continued to decline when the researchers checked at 11 months. In short, the pathology started to look like a neurodegenerative disease of aging such as FTD. The mice even showed inclusions positive for TDP-43 and ubiquitin. At the Vancouver meeting, Lewis encouraged other researchers with TDP-43 models to try the post-development variation.

TDP-43 Mice—Are They Good for Anything?
Clearly, there are plenty of differences between TDP-43 mice and human TDP-43 proteinopathies. “That does not mean the mice are useless,” Baloh said. “They can tell us pathways.” Elliott added that once researchers have a drug to test, it is worth trying it in a TDP-43 model. Given how many labs have generated and published TDP-43 mice by now, Elliott does not anticipate a much better model coming along.

Lutz advised that scientists fully understand the strains they are working with, and carefully choose questions the mice can accurately answer. For example, with Baloh’s model, the gut problem makes survival a poor outcome to measure. Yet researchers interested in other elements of disease, such as degeneration of neuromuscular junctions or how quickly the animals start dragging their limbs, might get just what they need out of this model.

To obtain robust results from Baloh’s model, Vieira cautioned scientists to stay away from the females. They live too long and their disease is too variable. Males could be useful—though not necessarily for ALS, he suggested; they might better model intestinal neuropathy. For his part, Vieira is still searching for the best TDP-43 model of ALS.

Whatever their worth as preclinical models, the TDP-43 and SOD1 mice are due to receive company. Researchers are working on animals transgenic for more recently discovered ALS genes such as fused in sarcoma and C9ORF72.—Amber Dance.

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Wegorzewska I, Bell S, Cairns NJ, Miller TM, Baloh RH. TDP-43 mutant transgenic mice develop features of ALS and frontotemporal lobar degeneration. Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18809-14. Epub 2009 Oct 15 PubMed.
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Arginine Methylation Distinguishes ALS-FUS From FTLD-FUS

21 Sep 2012

Some researchers have come to think of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) as two manifestations of the same pathology, but Christian Haass and Dorothee Dormann dissected out differences between the two in presentations at the 8th International Conference on Frontotemporal Dementias, held 5-7 September in Manchester, U.K. The scientists from Ludwig-Maximilians-Universität in Munich, Germany, also described their study in the EMBO Journal online September 11. They found that in cases of ALS due to FUS mutations, and frontotemporal lobar degeneration (FTLD) linked to FUS proteinopathy, the cellular mechanisms of disease are somewhat distinct. Both diseases feature FUS aggregates and stress granules, yet abnormal—aka subnormal—arginine methylation of FUS marks only FTLD, not ALS, pathology. Arginine methylation and subsequent defects in nuclear import might become a new area of investigation in neurodegenerative diseases, the researchers said.

In healthy cells, FUS is a nuclear protein. Via a nuclear localization sequence (NLS) on its carboxyl-terminal end, it hooks up with the nuclear shuttle transportin 1 to enter the nucleus. In some cases of ALS, however, mutations to the NLS render the protein cytosolic (ALS-FUS). There is also a subset of FTLD in which FUS is stuck in the cytoplasm (FTLD-FUS), but since its NLS is normal, something else must explain its mislocalization (see Urwin et al., 2010). Since FUS is known to be subject to post-translational methylation, Dormann, in Haass’ lab, investigated whether those methyl groups might influence its cellular address.

She started by treating HeLa cells expressing FUS NLS mutants with AdOx, a broad-spectrum methylation inhibitor. Despite having the mutation, FUS now traveled its normal path to the nucleus. The finding suggests that methylation powerfully influences FUS retention in the cytosol.

Thinking methylation might interfere with the transportin-FUS interaction, Dormann incubated recombinant transportin with either methylated or unmethylated synthetic FUS peptides. That confirmed that the methyls inhibit transportin binding to FUS; the unmethylated FUS peptides bound transportin most tightly. Further work revealed a new, large transportin-binding domain on FUS that is sensitive to arginine methylation, expanding scientists’ view of how transportin binds its cargo.

To investigate what this might mean to human disease, the team created antibodies specific for methylated FUS. In control brain sections, methylated FUS appeared only in the nucleus. In ALS cases, methylated FUS was present not only in the nucleus, but also in the cytoplasmic aggregates. In FTLD, methylated FUS was again nuclear—but the FUS-positive inclusions in the cytosol were unmethylated.

The different methylated states of the aggregated FUS suggest two different pathways for inclusion formation in ALS and FTLD, Haass said in his presentation. The ALS mechanism is fairly straightforward. Dormann and Haass posit that FUS’ defective NLS prevents it from binding transportin and sequesters it in the cytoplasm, where it accumulates and aggregates.

The situation is more complex in FTLD, the scientists suggest. In addition to the loss of FUS methylation Dormann discovered, previous studies have shown that when FUS aggregates in FTLD, it brings with it TAF15 and EWS, related members of the FET family of proteins, which are also subject to arginine methylation (see ARF related news story on Neumann et al., 2011). This trio shares transportin as a nuclear importer, and transportin itself also appears in the cytoplasmic inclusions in FTLD-FUS (Neumann et al., 2012). In this form of FTLD, therefore, it appears that FUS, TAF15, and EWS all suffer from some generalized nuclear import defect. All three, Haass and Dormann propose, are hypomethylated. With no methyl groups to interfere, they bind extra tightly to transportin and somehow drag it into cytoplasmic inclusions. FTLD-FUS is marked by a more generalized defect in transportin-mediated nuclear import, the authors suggest.

The work goes a long way toward solving a puzzle about ALS and FTLD, commented Emanuele Buratti of the International Centre for Genetic Engineering and Biotechnology in Trieste, Italy, in an e-mail to Alzforum. “Although ALS-FUS and FTLD-FUS were shown to have overlapping clinical phenotype and neuropathology, there was no explanation for why individuals affected by the same protein pathology developed one form of the disease as opposed to the other,” wrote Buratti, who was not involved in the study (see full comment, below).

“Although the mechanisms are different, the common feature, FUS intracellular mislocalization, supports the concept of ALS and FTLD being closely related, but not simply a single spectrum,” added Ian Mackenzie of Vancouver General Hospital in Canada, a study coauthor, in another e-mail.

The work is specific to ALS and FTLD with FUS pathology, Haass noted in an interview with Alzforum. Coauthor Manuela Neumann of the German Center for Neurodegenerative Diseases in Tübingen speculated that it might have broader significance as well. “Given that a lot of RNA binding proteins are arginine methylated, and [considering] the increasing number of RNA binding proteins involved in ALS and FTD, it is possible that dysregulation of arginine methylation might be involved also in non-FUSopathies,” she wrote in an e-mail to Alzforum (see full comment below).—Amber Dance

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Urwin H, Josephs KA, Rohrer JD, Mackenzie IR, Neumann M, Authier A, Seelaar H, van Swieten JC, Brown JM, Johannsen P, Nielsen JE, Holm IE, , Dickson DW, Rademakers R, Graff-Radford NR, Parisi JE, Petersen RC, Hatanpaa KJ, White CL, Weiner MF, Geser F, Van Deerlin VM, Trojanowski JQ, Miller BL, Seeley WW, van der Zee J, Kumar-Singh S, Engelborghs S, De Deyn PP, Van Broeckhoven C, Bigio EH, Deng HX, Halliday GM, Kril JJ, Munoz DG, Mann DM, Pickering-Brown SM, Doodeman V, Adamson G, Ghazi-Noori S, Fisher EM, Holton JL, Revesz T, Rossor MN, Collinge J, Mead S, Isaacs AM. FUS pathology defines the majority of tau- and TDP-43-negative frontotemporal lobar degeneration. Acta Neuropathol. 2010 Jul;120(1):33-41. PubMed.
Neumann M, Bentmann E, Dormann D, Jawaid A, Dejesus-Hernandez M, Ansorge O, Roeber S, Kretzschmar HA, Munoz DG, Kusaka H, Yokota O, Ang LC, Bilbao J, Rademakers R, Haass C, Mackenzie IR. FET proteins TAF15 and EWS are selective markers that distinguish FTLD with FUS pathology from amyotrophic lateral sclerosis with FUS mutations. Brain. 2011 Sep;134(Pt 9):2595-609. PubMed.
Neumann M, Valori CF, Ansorge O, Kretzschmar HA, Munoz DG, Kusaka H, Yokota O, Ishihara K, Ang LC, Bilbao JM, Mackenzie IR. Transportin 1 accumulates specifically with FET proteins but no other transportin cargos in FTLD-FUS and is absent in FUS inclusions in ALS with FUS mutations. Acta Neuropathol. 2012 Nov;124(5):705-16. PubMed.

C9ORF72 Steals the Show at Frontotemporal Dementia Meeting

21 Sep 2012

One year ago today, researchers uncovered the identity of a mysterious gene on chromosome 9 that had long been linked to frontotemporal dementia and amyotrophic lateral sclerosis. To no one’s surprise, C9ORF72 occupied center stage in dozens of presentations and posters at the 8th International Conference on Frontotemporal Dementias, held 5-7 September in Manchester, U.K. Attendees also discussed the other key genes and proteins that contribute to the disease, including tau, progranulin, TDP-43, and FUS, as featured in Alzforum coverage to come. In addition, scientists debated how to subdivide different types of frontotemporal dementia (FTD) and how amyotrophic lateral sclerosis (ALS) fits into the dementia spectrum.

Repeat expansions in C9ORF72, an open reading frame of unknown function, turned out to explain many cases of both diseases (see ARF related news story on Renton et al., 2011 and Dejesus-Hernandez et al., 2011). An open reading frame is a stretch of DNA between a start codon and a stop codon. As discussed at the meeting, scientists immediately tackled the gene from all angles. Clinicians began developing a consensus on how C9ORF72 disease presents itself. Cell biologists made early forays into understanding how a series of GGGGCC repeats contribute to pathology. And geneticists are working out the expansion’s origins and the rate at which it causes disease. Here is a summary of the state of C9ORF72 science at the conference.

Genetic Riddles
“C9ORF72 is a very frequent gene variant,” said Christopher Shaw, of King’s College London, during his presentation. Researchers reported that it explains from 20 to 67 percent of familial amyotrophic lateral sclerosis (ALS), depending on the population studied, making it the most prevalent genetic mutation in the disease. The expansion also appears in about 25 percent of familial frontotemporal dementia cases (FTD), as well as 7 percent of sporadic ALS and 5 percent of sporadic FTD (Majounie et al., 2012).

One challenge in understanding C9ORF72, Shaw noted, is that the expansion also shows up in 0.3 percent of the healthy population. “Incomplete penetrance is the rule,” he said. Based on families he has seen, Shaw estimated that by age 50, 9 percent of carriers will exhibit symptoms; by age 85, 74 percent would. Geneticists are particularly intrigued by the concept of genetic anticipation, whereby children become sick at a younger age than their parent did. This occurs in perhaps one-quarter of kindreds carrying the C9ORF72 variant; the age of onset appears to be about seven years earlier in children of carriers.

It may be that the repeat region grows in each successive generation, said Bradley Boeve of the Mayo Clinic in Rochester, Minnesota, in an interview with Alzforum. That will be challenging to prove. Brain tissue samples are hard to come by, and the only way to properly measure repeat size—which can range up into the thousands—is via Southern blotting. “That requires a lot of work,” commented Peter Heutink of the VU University Medical Centre in Amsterdam, The Netherlands, in his presentation.

The big step of the anticipation from one generation to the next puzzled some researchers at the meeting. “I do worry a little bit about that seven years,” said Bryan Traynor of the National Institute on Aging in Bethesda, Maryland. “Why isn’t this disease occurring in utero almost?” That is a good question, Boeve agreed. Boeve suggested to Alzforum that perhaps the age of onset does indeed reach a point in some families at which affected embryos do not survive, or that the disease manifests differently in young people.

Attendees had some fun speculating on the origin of the hexanucleotide expansion. Because people with the variant share a haplotype containing dozens of single-nucleotide polymorphisms in the region surrounding the C9 gene, many scientists suspect a single founder. Assuming 15 years per generation, that person might have lived around the time the Roman Empire fell, in the fifth century A.D., Traynor estimated. Since the mutation is most common in Scandinavian populations, he suggested—tongue in cheek—that perhaps Vikings were responsible for spreading it across the globe soon after (see ARF related news story on Ishiura et al., 2012 and Tsai et al., 2012). Shaw, who thought 15 years for each generation might be a bit short, calculated that C9ORF72 began expanding 6,300 years ago, but said it could have been any time between 2,700 and 16,500 years ago (Smith et al., 2012). Heutink suggested that the alternative to a single founder is that the shared haplotype itself is somehow predisposed to repeat expansions. It is possible that multiple massive expansions occurred independently, Shaw agreed in an e-mail to Alzforum.

Clinical Characteristics
Physicians could use answers to questions about penetrance and anticipation to better advise people who test positive for a C9 expansion and their families. At the same time, clinicians are learning to recognize signs of the C9 expansion before they even run a DNA sample.

“A lot of clinical descriptions have come forth…. It is clear that [the expansion] is highly heterogeneous in presentation, and it is difficult to distinguish FTD due to C9ORF72 mutations from FTD with other pathology,” wrote Robin Hsiung of the University of British Columbia in Vancouver, Canada, in an e-mail to Alzforum. “However, there are some features emerging that may alert the clinician to a C9ORF72 mutation,” added Hsiung. One such feature is a mixture of ALS and FTD in the family tree. Carriers can have either disease, or signs of both. Rigidity typical of parkinsonism provides another indicator. Approximately one-third of patients with the genetic expansion exhibit this rigidity, Boeve said.

People who present with ALS are more likely to have bulbar rather than limb onset when their ALS is due to C9ORF72 expansion. FTD is mostly of the “behavioral variant” subtype—people have executive dysfunction, apathy, and loss of empathy. People with FTD due to C9ORF72 are more likely than other FTD patients to exhibit “bizarre” beliefs and hallucinations, Boeve added (Snowden et al., 2012). For example, Boeve cares for patients who believe they are being spied upon or in danger from relatives. One believed he was infested by mites with a particular preference for his earlobe; another was convinced plastic bits were extruding from his scalp (Boeve and Graff-Radford, 2012).

Clinicians suspicious of C9 expansions will also find clues in brain images, conference speakers reported. Unlike FTD due to progranulin mutations, which tends to cause atrophy in a focused area on one side of the cortex, C9 pathology is widespread and symmetric between the two halves of the cortex and cerebellum. Spotting the hallmarks of the expansion in a clinical exam could save some money, in that physicians would order genetic tests only for the likeliest suspects, Boeve said

The Pathology Puzzler
One big question is how the C9ORF72 expansion damages the cell, and answers are beginning to come in. In a repeat of the perennial loss-of-function versus gain-of-function debate surrounding many neurodegenerative disease mutations, some scientists theorize that the extended repeats might prevent translation, depriving the cell of the protein’s as-yet unknown physiologic function. Others suggest that the extended RNA itself is toxic, perhaps due to aggregation. Boris Rogelj of the Josef Stefan Institute in Ljubljana, Slovenia, has begun to investigate the latter hypothesis. He presented his findings on the structure of the repeated nucleic acid in a poster.

Rogelj engineered up to four GGGGCC repeats in a DNA strand and examined the secondary structure by nuclear magnetic resonance and circular dichroism. He started with DNA because he worried an RNA might be unstable, but RNA studies are ongoing and thus far look similar to the DNA results, he told Alzforum. With the DNA, Rogelj found that the repeats tended to form a structure called a G-quadruplex. Four repeats line up side by side, as if they formed the four corners of a tall building. These kinds of structures are known to participate in regulating oxidative stress, Rogelj noted.

Researchers still need to discover the natural function of C9ORF72. Here, animal models could help. David Satelle of the University of Manchester told attendees about a C. elegans mutant he found in a standard worm collection. Called ok3062, the strain has a deletion in the nematode analogue of this open reading frame. Wild-type worms normally assume an “S” shape, but these mutants are “kinked” and swim at half the normal speed, Satelle said. These animals could be useful for screening drugs or genetic modifiers, he suggested.

A lighter question meeting attendees tossed around is how to shorten the clunky mouthful that is “C9ORF hexanucleotide intronic repeat expansion mutations.” Shaw abbreviated the phrase to “C9HIREM.” Members of Pam Shaw’s lab, at the Sheffield Teaching Hospital in the U.K., have started calling it the “Dallas mutation” because the telephone area code for the Texas city is 972. Others simply pronounce the gene name “corf” or “snorf.” Votes, anyone?—Amber Dance.

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Renton AE, Majounie E, Waite A, Simón-Sánchez J, Rollinson S, Gibbs JR, Schymick JC, Laaksovirta H, van Swieten JC, Myllykangas L, Kalimo H, Paetau A, Abramzon Y, Remes AM, Kaganovich A, Scholz SW, Duckworth J, Ding J, Harmer DW, Hernandez DG, Johnson JO, Mok K, Ryten M, Trabzuni D, Guerreiro RJ, Orrell RW, Neal J, Murray A, Pearson J, Jansen IE, Sondervan D, Seelaar H, Blake D, Young K, Halliwell N, Callister JB, Toulson G, Richardson A, Gerhard A, Snowden J, Mann D, Neary D, Nalls MA, Peuralinna T, Jansson L, Isoviita VM, Kaivorinne AL, Hölttä-Vuori M, Ikonen E, Sulkava R, Benatar M, Wuu J, Chiò A, Restagno G, Borghero G, Sabatelli M, ITALSGEN Consortium, Heckerman D, Rogaeva E, Zinman L, Rothstein JD, Sendtner M, Drepper C, Eichler EE, Alkan C, Abdullaev Z, Pack SD, Dutra A, Pak E, Hardy J, Singleton A, Williams NM, Heutink P, Pickering-Brown S, Morris HR, Tienari PJ, Traynor BJ. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron. 2011 Oct 20;72(2):257-68. Epub 2011 Sep 21 PubMed.
DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ, Nicholson AM, Finch NA, Flynn H, Adamson J, Kouri N, Wojtas A, Sengdy P, Hsiung GY, Karydas A, Seeley WW, Josephs KA, Coppola G, Geschwind DH, Wszolek ZK, Feldman H, Knopman DS, Petersen RC, Miller BL, Dickson DW, Boylan KB, Graff-Radford NR, Rademakers R. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron. 2011 Oct 20;72(2):245-56. Epub 2011 Sep 21 PubMed.
Majounie E, Renton AE, Mok K, Dopper EG, Waite A, Rollinson S, Chiò A, Restagno G, Nicolaou N, Simon-Sanchez J, van Swieten JC, Abramzon Y, Johnson JO, Sendtner M, Pamphlett R, Orrell RW, Mead S, Sidle KC, Houlden H, Rohrer JD, Morrison KE, Pall H, Talbot K, Ansorge O, , Hernandez DG, Arepalli S, Sabatelli M, Mora G, Corbo M, Giannini F, Calvo A, Englund E, Borghero G, Floris GL, Remes AM, Laaksovirta H, McCluskey L, Trojanowski JQ, Van Deerlin VM, Schellenberg GD, Nalls MA, Drory VE, Lu CS, Yeh TH, Ishiura H, Takahashi Y, Tsuji S, Le Ber I, Brice A, Drepper C, Williams N, Kirby J, Shaw P, Hardy J, Tienari PJ, Heutink P, Morris HR, Pickering-Brown S, Traynor BJ. Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study. Lancet Neurol. 2012 Apr;11(4):323-30. PubMed.
Ishiura H, Takahashi Y, Mitsui J, Yoshida S, Kihira T, Kokubo Y, Kuzuhara S, Ranum LP, Tamaoki T, Ichikawa Y, Date H, Goto J, Tsuji S. C9ORF72 Repeat Expansion in Amyotrophic Lateral Sclerosis in the Kii Peninsula of JapanC9ORF72 Repeat Expansion in ALS. Arch Neurol. 2012 Jun 4;:1-5. PubMed.
Tsai CP, Soong BW, Tu PH, Lin KP, Fuh JL, Tsai PC, Lu YC, Lee IH, Lee YC. A hexanucleotide repeat expansion in C9ORF72 causes familial and sporadic ALS in Taiwan. Neurobiol Aging. 2012 Sep;33(9):2232.e11-2232.e18. PubMed.
Smith BN, Newhouse S, Shatunov A, Vance C, Topp S, Johnson L, Miller J, Lee Y, Troakes C, Scott KM, Jones A, Gray I, Wright J, Hortobágyi T, Al-Sarraj S, Rogelj B, Powell J, Lupton M, Lovestone S, Sapp PC, Weber M, Nestor PJ, Schelhaas HJ, Asbroek AA, Silani V, Gellera C, Taroni F, Ticozzi N, Van den Berg L, Veldink J, Van Damme P, Robberecht W, Shaw PJ, Kirby J, Pall H, Morrison KE, Morris A, de Belleroche J, Vianney de Jong JM, Baas F, Andersen PM, Landers J, Brown RH, Weale ME, Al-Chalabi A, Shaw CE. The C9ORF72 expansion mutation is a common cause of ALS+/-FTD in Europe and has a single founder. Eur J Hum Genet. 2012 Jun 13; PubMed.
Snowden JS, Rollinson S, Lafon C, Harris J, Thompson J, Richardson AM, Jones M, Gerhard A, Neary D, Mann DM, Pickering-Brown S. Psychosis, C9ORF72 and dementia with Lewy bodies. J Neurol Neurosurg Psychiatry. 2012 Oct;83(10):1031-2. PubMed.
Boeve BF, Graff-Radford NR. Cognitive and behavioral features of c9FTD/ALS. Alzheimers Res Ther. 2012 Jul 20;4(4):29. PubMed.

TDP-43 Controls Blood Vessels in Fish, Is Phosphorylated in Worms

26 Sep 2012

The critters swim in rivers and squirm through the soil, and they pack a big punch in the lab. At the 8th International Conference on Frontotemporal Dementias, held 5-7 September in Manchester, U.K., attendees learned what zebrafish and nematodes, respectively, have to offer researchers studying TDP-43, a key pathological player in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Bettina Schmid and Christian Haass of the German Center of Neurodegenerative Diseases (DZNE) in Munich, Germany, knocked out the gene in zebrafish embryos and observed deformation not only in the animal’s motor neurons, but also muscles and blood vessels. Working with the roundworm Caenorhabditis elegans, Nicki Liachko of the Veterans Affairs Puget Sound Healthcare System in Seattle, Washington, identified the enzyme that phosphorylates TDP-43—an event that is key to the protein’s pathogenicity in the nematode and perhaps in people as well. She discovered the cell cycle kinase CDC7 is responsible for TDP-43 phosphorylation.

The researchers took advantage of the particular characteristics of their model system, Schmid noted in an interview with Alzforum. The RNA interference screens Liachko performed are easy to do in nematodes; moreover, unlike some other animal TDP-43 models, the worm actually mimics the TDP-43 phosphorylation seen in people. Zebrafish allowed Schmid to cleanly knock out TDP-43 and analyze stages of development that are difficult to study in mammals. Mouse embryos die before birth without TDP-43, for example, but that only tells researchers that the gene must be crucial for development. Fish embryos lacking TDP-43 die eight days after fertilization; however, since fish eggs are fertilized outside the mother’s body and develop into translucent embryos, Schmid was able to investigate why the knockouts expire.

Something Fishy
Zebrafish carry two homologues of the human TDP-43 gene, TARDBP and a “TARDBP-like” gene called TARDBPl. The former protein contains the two RNA recognition motifs and a carboxy-terminal glycine-rich domain found in human TDP-43, whereas the latter protein lacks the glycine domain. Mutations that cause ALS tend to cluster in that region. When the researchers knocked out one or the other fish gene, “the result was really boring,” complained Haass in his talk. Nothing seemed to happen to the motor neurons or any other part of the fish embryo.

At first, the researchers, assuming that the glycine-rich domain was crucial to TDP-43 function, were surprised that fish lacking TARDBP were healthy. But looking more closely at the sequence of the TARDBP-like protein gene, they saw a potential answer. The glycine-rich code was present, but usually spliced out. A Western blot showed that the glycines were included in TARDBPl when TARDBP was not present. “This glycine-rich domain is so extremely important for the function that the fish makes a backup version,” Schmid concluded.

To obtain a full TDP-43 knockout phenotype, then, Schmid had to delete both TARDBP and TARDBPl genes. In these embryos, Schmid saw shortened motor neuron axons, as might be expected from deleting a gene linked to ALS. Furthermore, she saw that muscles degenerated and red blood cells pooled in the embryo’s yolk instead of coursing through the circulatory system. Blood vessels were wildly disorganized in the brain and body of the double mutant. “Probably TDP-43 is, at least developmentally, required to maintain muscles, the vasculature, and axonal outgrowth,” Haass concluded. Schmid suggested that researchers studying ALS and FTD in people might find it fruitful to look for muscle and blood vessel defects.

Aaron Gitler of Stanford University, who was not involved in the study, noted that this is the first vertebrate animal model with TDP-43 genetically removed from the genome—not eliminated by RNA interference, which tends to leave some of the protein around. Even if the defects in vasculature and musculature turn out to be specific to fish, studying the affected cells could still inform researchers about TDP-43 pathology that might occur in human neurons, Gitler added.

Worm Work
Liachko, who works in the laboratory of Brian Kraemer at the University of Washington in Seattle, studies worms that express mutant human TDP-43. Their motor neurons degenerate, and they are “severely uncoordinated,” Liachko said in her presentation. The worms tend to coil up and rarely stray from where they hatched. She previously reported that phosphorylation at serines 409 and 410 contributes to the toxicity of mutant TDP-43 in C. elegans (Liachko et al., 2010). This contrasts with some mouse TDP-43 models, which have not exhibited the same post-translational modification, although some newer models do, Liachko told Alzforum.

Where there is phosphorylation, there must be a kinase, and Liachko described her search for it. Using short interfering RNAs, she knocked down 186 different enzymes in worms with mutant TDP-43, looking for animals that moved normally. She found 12 that did, and checked whether the silenced genes directly affected TDP-43. In three hits, phosphorylation of the protein fell.

The candidates were the homologues of human CDC7, TTBK1, and TTBK2. Pairing the purified human kinases with TDP-43 in vitro, Liachko found that only CDC7 directly phosphorylated TDP-43. Being a cell cycle regulator, CDC7 is not an enzyme scientists would expect to find active in non-dividing neurons. Even so, Liachko showed that the kinase appears in the cell bodies and nuclei of neurons in the human brain. CDC7 clearly has functions beyond the cell cycle, Liachko said, although she does not know what they might be.

Could inhibiting CDC7 prevent TDP-43 toxicity? Using the small molecule inhibitor PHA767491, Liachko reduced not only TDP-43 phosphorylation, but also neurodegeneration in her C. elegans model. The drug also limited TDP-43 phosphorylation in mouse NSC-43 cells, which are a hybrid motor neuron-neuroblastoma line where TDP-43 phosphorylation does occur.

CDC7 inhibitors were tested in clinical trials to treat cancer. They interfere with the tumor cells’ cycling, causing apoptosis. Liachko noted that attacking a crucial cell cycle protein is one thing in acute chemotherapy with unpleasant side effects, but quite another in progressive neurodegenerative diseases such as ALS and FTD. “You cannot chronically downregulate CDC7 in a person and expect a good outcome,” she said. But based on her data, she believes, “the idea of preventing TDP-43 phosphorylation is promising.”

Schmid told Alzforum the study was elegant and worthy of follow-up. It demonstrates the value of screens in model systems, Gitler added. Both scientists suggested the next step should be to analyze TDP-43 phosphorylation in rodent models. “We will be able to do that soon,” Liachko said, since now there are mice that recapitulate the phosphorylation phenotype.—Amber Dance.

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Can Epigenetics Explain Variable Progranulin Expression?

28 Sep 2012

Researchers at the 8th International Conference on Frontotemporal Dementias, held 5-7 September in Manchester, U.K., got something new to think about with respect to how the cell controls expression of the disease-linked gene progranulin. Julia Strathmann of the German Center of Neurodegenerative Diseases (DZNE) in Munich explained how methylation of progranulin’s promoter turns down the gene. “We have identified new insights into how progranulin expression can be controlled, namely, by DNA methylation,” Strathmann wrote in an e-mail to Alzforum. Drugs that remove those methyl groups might boost progranulin and thus treat the disease, she suggested in an interview.

Strathmann’s talk received an award for the best oral presentation by a young researcher at the Manchester conference. Methylation was a hot topic at the meeting; speakers described how a different kind of methyl modification—to proteins—causes mislocalization of FUS, another dementia-linked protein (see ARF related news story on Dormann et al., 2012).

People carrying certain progranulin mutations, who suffer frontotemporal lobar degeneration (FTLD), have lower-than-normal concentrations of progranulin in their blood (Sleegers et al., 2009). However, “the physiological regulation of progranulin expression in the nervous system remains largely unexplored,” noted Louis De Muynck of VIB Leuven, Belgium, in an e-mail to Alzforum. De Muynck attended the meeting but was not involved in the study. Strathmann’s work begins to chart that new territory.

Among healthy people, and even those with sporadic FTLD, progranulin concentration in the blood differs measurably. One study found that those with the wild-type gene had anywhere from 125 to 375 nanograms of the protein per milliliter of plasma, regardless of FTLD status (Finch et al., 2009). Strathmann, who earned her graduate degree in an epigenetics lab and now works with Dieter Edbauer, followed a hunch that methylation might influence progranulin expression. Methyl groups, attached to cytosine- and guanine-rich sequences, often suppress genes.

Strathmann examined lymphoblasts obtained from several healthy controls, as well as from two patients with progranulin missense mutations. The amount of progranulin mRNA present and the concentration of the protein secreted into the culture media differed among the cells. By quantitative mass spectrometry, she determined that methylation of the progranulin promoter explained the pattern. There was a textbook inverse correlation, she said. The more methylated the promoter, the less progranulin the cell made. Using a drug called decitabine, which blocks DNA methyltransferases, she was able to boost progranulin mRNA and protein in the lymphoblasts.

What about in the brains of people with FTLD? Strathmann, in collaboration with Christine Van Broeckhoven of the University of Antwerp, Belgium, measured progranulin promoter methylation in brain tissue from five healthy controls and 10 people who had FTLD, including four people with progranulin mutations, two with mutations in another FTLD gene, VCP, and four with sporadic disease. The researchers observed a “small but significant” increase in progranulin promoter methylation in all FTLD cases, Strathmann said. There was also a trend toward lowered progranulin mRNA in the brain samples from FTLD cases, Strathmann wrote in an e-mail, but she was unable to examine progranulin protein levels in this tissue.

Researchers led by Daniela Galimberti at the University of Milan, Italy, recently found the same link between progranulin methylation and expression (Galimberti et al., 2012). Amid white blood cells from 38 people with sporadic FTLD, the progranulin promoter was methylated at 62 percent of possible sites, on average, compared to 46 percent among controls. “The methylation likely leads to a decreased expression of progranulin,” Galimberti wrote. She also observed a trend toward less progranulin in the FTLD cases, and a correlation between methylation and expression, but this was not statistically significant. In addition, the Milan researchers observed a wide range of methylation values and plenty of overlap between patients and controls.

“This hypermethylation is probably not the sole cause of why these people developed FTLD,” Strathmann wrote in an e-mail. In contrast, progranulin levels clearly distinguish those with mutations in the gene from sporadic FTLD and healthy controls (Finch et al., 2009). Thus, the loss of progranulin due to mutations is a much stronger effect than the downregulation by methylation, making mutation status a better marker for disease, Strathmann wrote.

Strathmann also tested brain samples for expression of methyltransferases by quantitative reverse transcriptase polymerase chain reaction. She found that the FTLD cases had more of the mRNAs for the DNA methyltransferases Dnmt1 and Dnmt3a, compared to controls. Since these enzymes modify many targets, Strathmann now plans to look for altered epigenetic patterns across the genome in FTLD.

“This study provides us with important new insights into how progranulin expression is regulated,” commented Peter Heutink of the VU University Medical Centre in Amsterdam, The Netherlands, in an e-mail to Alzforum. Heutink, who was not involved in the research, also noted it raises new questions. Is the methylation pattern set, or dynamic over time? Might other epigenetic factors, such as histone modification, affect progranulin expression? And finally, can scientists alter the methylation? Attacking the epigenetic pattern would be an addition to other progranulin therapy ideas, such as preventing progranulin endocytosis (see ARF related news story) or administering small molecules that amp up its production, as in the work of De Muynck and others (see ARF related news story).—Amber Dance.

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Dormann D, Madl T, Valori CF, Bentmann E, Tahirovic S, Abou-Ajram C, Kremmer E, Ansorge O, Mackenzie IR, Neumann M, Haass C. Arginine methylation next to the PY-NLS modulates Transportin binding and nuclear import of FUS. EMBO J. 2012 Sep 11; PubMed.
Sleegers K, Brouwers N, Van Damme P, Engelborghs S, Gijselinck I, van der Zee J, Peeters K, Mattheijssens M, Cruts M, Vandenberghe R, De Deyn PP, Robberecht W, Van Broeckhoven C. Serum biomarker for progranulin-associated frontotemporal lobar degeneration. Ann Neurol. 2009 May;65(5):603-9. PubMed.
Finch N, Baker M, Crook R, Swanson K, Kuntz K, Surtees R, Bisceglio G, Rovelet-Lecrux A, Boeve B, Petersen RC, Dickson DW, Younkin SG, Deramecourt V, Crook J, Graff-Radford NR, Rademakers R. Plasma progranulin levels predict progranulin mutation status in frontotemporal dementia patients and asymptomatic family members. Brain. 2009 Mar;132(Pt 3):583-91. PubMed.
Galimberti D, D'Addario C, Dell'osso B, Fenoglio C, Marcone A, Cerami C, Cappa SF, Palazzo MC, Arosio B, Mari D, Maccarrone M, Bresolin N, Altamura AC, Scarpini E. Progranulin gene (GRN) promoter methylation is increased in patients with sporadic frontotemporal lobar degeneration. Neurol Sci. 2012 Jul 14; PubMed.

Are TDP-43 Mice Living Up to Expectations?

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Arginine Methylation Distinguishes ALS-FUS From FTLD-FUS

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C9ORF72 Steals the Show at Frontotemporal Dementia Meeting

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TDP-43 Controls Blood Vessels in Fish, Is Phosphorylated in Worms

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Can Epigenetics Explain Variable Progranulin Expression?

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