Exploring Genome Fragmentation in Neurodegeneration

22 Feb 2013

Researchers gathered in Santa Fe, New Mexico, 4-7 February 2013 for the Keystone Symposium “New Frontiers in Neurodegenerative Disease Research.” Whether sipping wine in the poster hall or mingling at a concurrent symposium on neurogenesis, scientists who study different conditions found common ground in their focus on decaying neurons. The changing genome stood out as an emerging theme, noted co-organizer Li-Huei Tsai of MIT. Researchers are looking beyond the simple sequence of inherited genes to what physically happens to those genes throughout life. In one session, Tsai and other researchers discussed the disintegration of the genome in degenerating neurons. Single- and double-stranded breaks in DNA appear in neurodegenerative diseases, including Alzheimer’s, amyotrophic lateral sclerosis (ALS), and the childhood neurodegenerative syndrome ataxia telangiectasia. Moreover, key proteins in those diseases—amyloid precursor protein (APP), fused in sarcoma (FUS), and ataxia telangiectasia mutated (ATM), respectively—somehow influence DNA repair or the response to DNA damage, speakers reported.

Neurons confront a unique challenge, Tsai said in her presentation, because they don't divide, and therefore must keep their DNA whole. At the same time, neurons are subject to a barrage of reactive oxygen species that chemically nick DNA strands. “Our genome is constantly attacked,” Tsai told Alzforum. Because neurons are so essential, they must survive even if their DNA is damaged. “The inability to replace these cells puts a mandate on their preservation,” said speaker Bruce Yankner of Harvard Medical School. Young neurons repair DNA damage, but neurons beset by old age, mutations, or other disease risk factors cannot keep pace with the mounting injuries, Tsai suggested. “There is growing evidence supporting the idea that DNA damage is an early event in neurodegenerative disease,” commented Eric Huang of the University of California, San Francisco, who attended the meeting. “Evidence of DNA damage can be detected in neurons in AD, PD, and ALS patients,” he said.

AD Protein Blocks DNA Damage Response
Yankner has found that as people age, they accumulate oxidative DNA damage, particularly in the hippocampus (see ARF related news story on Lu et al., 2004). In AD brains, genomic blemishes such as double-stranded breaks are even more prevalent than in normal brains, Yankner said.

Does this DNA damage contribute to aging and AD? To investigate the effects of DNA damage, Yankner’s team worked with mice lacking the gene for the DNA repair protein XRCC4 in their neurons and glia. The mice accumulate double-strand DNA breaks with age, but show no signs of neurodegeneration. They do, however, lose dendritic spines, particularly in the hippocampus. Yankner and Tsai collaborated to study synaptic plasticity in brain slices from these animals via electrophysiology. They discovered that the mutants were unable to sustain long-term potentiation—a marker of synaptic strengthening. The mice struggled to find the submerged platform in a water maze test of spatial memory. Unrepaired DNA damage pushes the brain towards faulty synaptic activity and cognition, Yankner said.

How might that relate to AD pathology? To find out, the researchers crossed their XRCC4-null mice with the J20 APP model. The double mutants accumulated more DNA damage than the XRCC4 mutants, particularly in glia. They also lost neurons, mostly in the hippocampus, some in the cortex, and none in the cerebellum. This pattern is reminiscent of human AD pathology, Yankner said. He suggested that an APP mutation plus DNA damage might better reproduce the etiology of AD than the APP model alone. DNA damage due to the XRCC4 deletion might mimic the decades of attacks on DNA that are endured by older people but not short-lived mice.

How does mutant APP exacerbate faulty DNA repair? J20 mice accumulate Aβ, widely accepted as the toxic entity in these models, but it does not seem to be the culprit here. Introducing a third mutation—loss of the Aβ-generating enzyme BACE1—did not relieve the phenotype. Yankner concluded that it was the mutations in APP itself, not the excess of processed amyloid-β fragments, that boosted DNA damage in the XRCC4/J20 double mutants. From that, he inferred that wild-type APP plays some role in DNA damage or repair, or in the cellular response to that damage.

Hints to what that role might be came when the scientists analyzed genes expressed in the hippocampus of XRCC4-negative animals. They observed upregulation of a p53-led apoptosis pathway as well as neuroinflammation. Based on this observation, Yankner proposed a hypothetical model whereby full-length APP normally tempers the p53 damage response, preventing apoptosis in neurons and allowing them to still function or repair the damage. APP mutations reduce this protection and the neurons die.

If Yankner’s model is correct, researchers suggested during the question-and-answer period, then crossing the XRCC4-negative mice with an APP knockout should also produce mice with DNA damage and neurodegeneration, just like the J20/XRCC4 double mutants. Yankner’s group is pursuing that idea now, he said.

Faulty Genome Repair in ALS
In previous studies of the DNA damage response, Tsai found that histone deacetylase 1 (HDAC1) promotes repair of double-strand breaks (see ARF related news story on Kim et al., 2008). To find other participants in that process, her group immunoprecipitated HDAC1 and associated proteins from mouse brain. To their surprise, one partner was FUS, which is mutated in familial ALS (see ARF related news story on Kwiatkowski et al., 2009, andVance et al., 2009). As it turns out, researchers many years ago had discovered that chromosomes in mice lacking FUS are unstable (Hicks et al., 2000).

The Tsai team found that cultured cells rapidly recruited FUS to sites of DNA damage. Challenging primary neurons with DNA-breaking chemicals enhanced the HDAC1-FUS interaction. Knocking down FUS in cell lines reduced DNA repair to almost nil, and ALS-linked mutations in FUS also impaired DNA repair and HDAC1 binding, Tsai said.

Tsai collaborated with Huang to check if the results hold up in vivo. In a mutant FUS mouse his group bred, more DNA appeared damaged than in normal mice. The same occurs in people, it appears. The researchers observed more DNA damage in motor cortex neurons from a person who died of ALS caused by the same FUS mutation. “There is no doubt that human ALS patients harboring this mutation show profound DNA double-stranded breaks in their neurons,” Tsai said. This could be a factor contributing to neurodegeneration, with damage accumulating over a lifetime until neurons can no longer sustain their fragmented genome, she suggested.

“I thought her study clearly demonstrated that FUS and potentially other ALS-associated proteins have a critical role in the DNA damage response, thus providing novel mechanistic understanding for how this protein causes disease, as well as opportunities for therapeutic intervention,” wrote Todd Cohen of the University of Pennsylvania in Philadelphia, who attended the meeting, in an e-mail to Alzforum.

DNA Lesions Build Up in Childhood Neurodegeneration
Peter McKinnon of St. Jude Children’s Research Hospital in Memphis, Tennessee, discussed not a disease of aging, but one of childhood. Despite its early onset, ataxia telangiectasia (AT) represents a “hallmark example” of a neurological disease caused by genomic instability, McKinnon said. This rare condition results from mutations in the kinase ATM, which attends to double-stranded DNA breaks. The disease affects multiple organs including the brain, blood vessels, and immune system, causing symptoms such as unsteady movement, cancer, and sensitivity to X-rays. Children are typically wheelchair-bound by the time they are five or six, McKinnon said. Progressive neurodegeneration starts in the cerebellum and spreads throughout the nervous system.

McKinnon proposed that the problem in children with AT is an overabundance of DNA lesions bound to the enzyme topoisomerase 1. The isomerase unwinds supercoiled DNA, opening it up for transcription or replication. It acts by making a single-strand nick, so the DNA can untwist. Before that nick can be repaired, topoisomerase must vacate the spot, but in ATM-deficient mice, McKinnon’s group found that topoisomerase stuck fast to the genomic DNA. This occurred despite normal overall concentrations of topoisomerase in the mutant animals.

McKinnon proposed that ATM normally prevents buildup of the enzyme topoisomerase on chromosomes. Without ATM, the isomerase sticks to nicked DNA. It not only prevents its repair, but also physically blocks transcription of nearby genes and interferes with DNA replication. The latter is particularly important during development of the nervous system, when cell division drives growth. When the DNA replication machinery hits one of those single-strand breaks, it could stall, truncating the daughter chromosome. That damage would promote apoptotic signals and cell death, McKinnon suggested.

More generally, unrepaired DNA could lead to all sorts of problems such as mutations and altered transcriptional patterns, Huang suggested. While the precise mechanisms linking damaged DNA to cell degeneration remain uncertain, genomic damage appears to play a role in several neurodegenerative conditions. “During aging, perhaps the [DNA repair] machinery gets worn out, just as reactive oxygen species are ramping up,” Tsai said.—Amber Dance.

References

News Citations

Paper Citations

Lu T, Pan Y, Kao SY, Li C, Kohane I, Chan J, Yankner BA. Gene regulation and DNA damage in the ageing human brain. Nature. 2004 Jun 24;429(6994):883-91. PubMed.
Kim D, Frank CL, Dobbin MM, Tsunemoto RK, Tu W, Peng PL, Guan JS, Lee BH, Moy LY, Giusti P, Broodie N, Mazitschek R, Delalle I, Haggarty SJ, Neve RL, Lu Y, Tsai LH. Deregulation of HDAC1 by p25/Cdk5 in neurotoxicity. Neuron. 2008 Dec 10;60(5):803-17. PubMed.
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.
Hicks GG, Singh N, Nashabi A, Mai S, Bozek G, Klewes L, Arapovic D, White EK, Koury MJ, Oltz EM, Van Kaer L, Ruley HE. Fus deficiency in mice results in defective B-lymphocyte development and activation, high levels of chromosomal instability and perinatal death. Nat Genet. 2000 Feb;24(2):175-9. PubMed.

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Up-and-Coming ALS Mice Leave Scientists ConFUSed

25 Feb 2013

Mouse engineers presented the latest models overexpressing the amyotrophic lateral sclerosis gene fused in sarcoma (FUS) at “New Frontiers in Neurodegenerative Disease Research,” a Keystone Symposium held 4-7 February 2013 in Santa Fe, New Mexico. These animals have been four years in the making. This is a long time even for mice (see ARF related news story). Part of the challenge was that the protein appears to regulate its own expression, so it was difficult to ramp it up to abnormally high levels, said Shuo-Chien Ling of the University of California, San Diego. Ling presented his model on a poster. Eric Huang of the University of California, San Francisco, introduced his new mice. These strains only partly recapitulate the pathology seen in human disease or other ALS models, dying young with moderate neurodegeneration. Scientists at the meeting puzzled over how they relate to human disease.

Overall, FUS transgenic mice developed by multiple research groups exhibit signs of age-dependent, progressive motor neuron disease akin to ALS, such as difficulty moving, faulty coordination, muscle wasting, paralysis, and early death. Ling also found that pathology was dose dependent. Mild disease ensued from one FUS transgene, while severe symptoms developed in mice with two copies. Both teams observed that mutant FUS caused worse symptoms than wild-type, although the normal protein was sufficient to cause disease when expressed at high levels. This matches well with previous rat and fruit fly models, commented Udai Pandey of the Louisiana State University Health Sciences Center in New Orleans, who was not involved in the presented research. Together, the new and the old models suggest that FUS gains a toxic function when mutated, he said (Verbeeck et al., 2012; Huang et al. 2012; Xia et al., 2012).

Huang’s mice express wild-type human FUS or an arginine-521-cysteine (R521C) mutation linked to human disease. By one to three months of age, their motor coordination began to suffer, their muscles were wasting away, and their neuromuscular junctions were losing their innervation. Most of the animals died a few weeks after symptoms started. However, Huang and colleagues were surprised to see the mice retained more neurons than typically seen in ALS models. At the end stage of disease they still possessed half the motor neurons normally found in the anterior horn, whereas in ALS models expressing mutant human superoxide dismutase 1 (SOD1), 90 percent of those neurons are gone at the end of life, Huang said.

To investigate this discrepancy, Huang collaborated with Steven Finkbeiner of the Gladstone Institutes in San Francisco. The researchers cultured neurons from the mice and confirmed that FUS expression was less toxic than other proteins implicated in neurodegeneration, such as TDP-43 or huntingtin. They did see stunted dendrites in transgenic neurons expressing either mutant or wild-type FUS. Going back to the mice, the researchers stained tissue and saw stubby dendrites in the sensorimotor cortex and cervical spinal cord. This synaptic defect might explain the mice’s symptoms even if their neurons survive, suggested the researchers.

How do Ling’s mice compare? They express wild-type FUS or the ALS-linked mutations arginine-514-guanine or R521C. These mice also retained more motor neurons than do other models. Although their motor control declined with age, they lived for a year or more. Limited FUS overexpression may make their disease mild, Ling said. Ling found that the human transgene dampened production of endogenous mouse FUS, keeping levels relatively low overall. Homozygotes with two copies of the human gene expressed about one and a half times the normal amount of FUS. Disease progressed faster in those animals. They had widespread neurodegeneration, losing about a third of their motor neurons and dying at 40 days of age.

Modeling ALS

The results jibe with a recently published mouse model expressing wild-type human FUS (Mitchell et al., 2012). Those authors also observed that FUS transgenes turned down endogenous FUS production, and that two transgene copies were necessary to produce disease. And here, too, 40 percent of motor neurons remained in the lumbar spinal cord even though homozygous mice died by 12 weeks of age.

What, then, do these mice tell researchers about the mechanism of FUS-based disease? In coimmunoprecipitation experiments, Huang observed FUS complexes. “The mutant protein has a higher propensity to form aggregates with itself and also with the endogenous wild-type protein,” he said. He proposed it could sequester normal FUS from its interactions with mRNA. Huang and Ling both observed mRNA splicing defects in their animals, and Ling’s homozygous FUS mutants showed splicing patterns similar to FUS knockdowns. Ling also saw accumulation of p62, which indicates blocked autophagy pathways.

Ling suggested that FUS normally ensures RNA is properly processed. Altering FUS homeostasis would result in both a loss of this normal splicing function and a gain of toxic function, because excess FUS inhibits autophagy. This latter angle opens up a potential treatment strategy, Pandey noted, because the drug rapamycin induces autophagy. An immunosuppressant used to prevent transplant rejection, rapamycin is also under study as a potential cancer drug.

Ling said no new mouse models for ALS fully mimic the human condition. Neither he nor Huang saw the widespread neurodegeneration and cytoplasmic FUS aggregates typical of the proteinopathy in people. Researchers modeling TDP-43 proteinopathy have made similar observations (see ARF related news story), and, of course, researchers in Alzheimer’s and Parkinson’s have for many years had to make do with partial models. ALS researchers have been “spoiled” by the SOD1 mouse, Ling suggested, with its rapid and severe disease resulting from one point mutation. Unfortunately, SOD1 mutations represent a tiny sliver of all ALS cases. For FUS and TDP-43 mice to better mimic human symptoms, they might require a second hit. “We need to make much more thorough models,” he said.—Amber Dance.

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References

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Paper Citations

Verbeeck C, Deng Q, Dejesus-Hernandez M, Taylor G, Ceballos-Diaz C, Kocerha J, Golde T, Das P, Rademakers R, Dickson DW, Kukar T. Expression of Fused in sarcoma mutations in mice recapitulates the neuropathology of FUS proteinopathies and provides insight into disease pathogenesis. Mol Neurodegener. 2012;7:53. PubMed.
Huang C, Tong J, Bi F, Wu Q, Huang B, Zhou H, Xia XG. Entorhinal cortical neurons are the primary targets of FUS mislocalization and ubiquitin aggregation in FUS transgenic rats. Hum Mol Genet. 2012 Nov 1;21(21):4602-14. PubMed.
Xia R, Liu Y, Yang L, Gal J, Zhu H, Jia J. Motor neuron apoptosis and neuromuscular junction perturbation are prominent features in a Drosophila model of Fus-mediated ALS. Mol Neurodegener. 2012;7:10. PubMed.
Mitchell JC, McGoldrick P, Vance C, Hortobagyi T, Sreedharan J, Rogelj B, Tudor EL, Smith BN, Klasen C, Miller CC, Cooper JD, Greensmith L, Shaw CE. Overexpression of human wild-type FUS causes progressive motor neuron degeneration in an age- and dose-dependent fashion. Acta Neuropathol. 2012 Sep 9; PubMed.

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Does ALS Gene Police RNA, Keep Strands From Entangling?

28 Feb 2013

Like a knot in your shoelace, entwined mRNAs can interfere with the nucleic acid’s normal actions. In amyotrophic lateral sclerosis, could it be that the cell overreacts to excess RNA snarls by responding with a toxic anti-virus response? Tassa Saldi of the University of Colorado in Boulder put forth this theory at the Keystone Symposium “New Frontiers in Neurodegenerative Disease Research,” held 4-7 February 2013 in Santa Fe, New Mexico. She studied the Caenorhabditis elegans orthologue of the ALS gene TDP-43, called TDP-1. Unlike TDP-43-negative mammals, C. elegans lacking TDP-1 survive, allowing her to examine the downstream effects on mRNAs.

“Their study was a great example of the importance of simple model organisms in learning about the normal function of disease proteins,” commented Aimee Kao of the University of California, San Francisco, in an e-mail to Alzforum. “It will hopefully stimulate some new thinking about the function of TDP-43 and how mutations cause disease.”

TDP-43 regulates expression and splicing of thousands of RNAs. To understand TDP-1’s role in the nematodes, Saldi, who works in the laboratory of Christopher Link, sequenced the transcriptome of the deletion strain. She discovered that 1,200 transcripts were over- or underexpressed compared to normal worms, and 350 genes were differentially spliced. No major gene categories emerged from the gene set, however, leaving few hints as to TDP-1’s primary effects.

Going through the list of genes one by one, Saldi did discover a common theme. It was gene overlap, a phenomenon where a given nucleotide sequence in one gene is also expressed as part of another gene (Sanna et al., 2008). Many genes differentially regulated in TDP-1’s absence overlapped with other genes, and the common sequences ran in opposite directions. This could occur if genes on opposite DNA strands share antiparallel coding sequences. It could also happen when one gene’s very long intron contains a second gene. This is not the first time long introns have been linked to TDP pathology; extended introns in the mouse genome are among the top targets of TDP-43 activity (see ARF related news story on Polymenidou et al., 2011, and Tollervey et al., 2011).

In general, about 8 percent of the worm genome overlaps, Saldi said. In the 1,550 genes that depend on TDP-1 for proper regulation, 35 to 45 percent were overlappers. The dataset also contained an unusually high number of introns of three kilobases or longer.

How does the TDP-1 knockout affect overlapping genes? If both sides of the DNA are transcribed at the same time, then their mRNAs are at risk of annealing to form a double-stranded structure. In fact, loss of TDP-1 resulted in noticeable dsRNA buildup in the worms, Saldi found. She stained the animals or individual tissues with an antibody to dsRNA and observed large nuclear inclusions. She could clear those aggregates by adding double-strand-specific RNase, but not RNase for single-stranded RNA, confirming their double-stranded structure.

Double-stranded RNAs, labeled with an antibody (red), accumulate in the nuclei of C. elegans lacking TDP-1. Image courtesy of Tassa Saldi, University of Colorado, Boulder

Saldi hypothesized that TDP-1 might work in RNA editing, which has evolved to disrupt these dsRNAs. Adenosine deaminases swap adenines for inosines, which pair awkwardly with the guanine on the opposite strand, forcing the dsRNA to unwind. This repair typically happens in untranslated regions or introns, and so does not interfere with the protein code, Saldi said.

When Saldi examined the editing system in her animals, she observed that the adenine-to-inosine transition still occurred in the transcriptome of TDP-1-negative worms. In fact, the deletion strain had more inosines than normal. Therefore, the editing process is working properly, Saldi said. She suspects that TDP-1 normally acts upstream, destabilizing dsRNAs so they unwind without editing. Worms lacking TDP-1, then, would require extra editing. Another possibility, she added, is that TDP-1 participates in dsRNA degradation.

Worms, of course, are not people, but Saldi found hints that TDP-43 in human cells performs a similar function. When she knocked down TDP-43 in HeLa cervical cancer cultures, dsRNA inclusions formed in the nucleus. Similarly, dsRNA inclusions formed in TDP-43-deficient M17 neurons, but this time the inclusions were cytoplasmic.

How might dsRNA aggregates contribute to neurodegeneration? Double-stranded RNA in the cytoplasm should raise a red flag, said Saldi, because it could be evidence of infection with a dsRNA virus. A natural response would be the interferon immune pathway that fights infection, but this can also be toxic. Could that be how TDP loss damages neurons? Supporting this theory, Saldi noted that many of TDP-43’s mRNA targets are involved in the interferon response (Polymenidou et al., 2011).—Amber Dance.

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References

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Sanna CR, Li WH, Zhang L. Overlapping genes in the human and mouse genomes. BMC Genomics. 2008;9:169. PubMed.
Polymenidou M, Lagier-Tourenne C, Hutt KR, Huelga SC, Moran J, Liang TY, Ling SC, Sun E, Wancewicz E, Mazur C, Kordasiewicz H, Sedaghat Y, Donohue JP, Shiue L, Bennett CF, Yeo GW, Cleveland DW. Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Nat Neurosci. 2011 Apr;14(4):459-68. PubMed.
Tollervey JR, Curk T, Rogelj B, Briese M, Cereda M, Kayikci M, König J, Hortobágyi T, Nishimura AL, Zupunski V, Patani R, Chandran S, Rot G, Zupan B, Shaw CE, Ule J. Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nat Neurosci. 2011 Apr;14(4):452-8. PubMed.

Does New Amyloid Protein Lend Tau a Hand?

01 Mar 2013

The tangled protein tau does not stand alone in aggregates. In people with Alzheimer’s and AD model mice, tau appears to have a partner in EF Hand 2 (EFhd2), according to work presented by the laboratory of Irving Vega, of the University of Puerto Rico in Rio Piedras, at the Keystone Symposium "New Frontiers in Neurodegenerative Disease Research," held 4-7 February 2013 in Santa Fe, New Mexico. Yancy Ferrer-Acosta reported on results from her recent publication in the January 18 Journal of Neurochemistry online, in which she found that EFhd2 forms fibrils in vitro and granules in brain tissue from people with Alzheimer’s. EFhd2 behaves like an amyloid, she concluded. And like its binding partner tau, EFhd2 undergoes phosphorylation by the Cdk5/p25 complex, as Edwin Vázquez-Rosa described in another poster. Finally, in an effort to understand EFhd2’s normal role, Eva Rodríguez-Cruz in the lab is closely watching the development of EFhd2 knockout mice. They have not yet shown a phenotype, but may do so with age, she reported at the meeting.

"[Irving] has done a very interesting series of studies in human AD and tauopathy mouse models which I think justifies his conclusions that … EF Hand 2 is a novel amyloid protein associated with tau," commented John Trojanowski of the University of Pennsylvania, Philadelphia, in an e-mail to Alzforum. However, Trojanowski cautioned that tau tangles trap many bystander proteins, so subsequent research should determine if EFhd2’s association with tau contributes to neurodegeneration.

Vega has been studying EFhd2 since 2008, when he discovered it coimmunoprecipitated with tau isolated from the brains of JNPL3 tauopathy model mice. EFhd2 appeared throughout the body, with the highest levels in the brain. It associated with tau only in aging, sickly animals, not in young or healthy ones, and Vega found the same tau-EFhd2 partnership in brain tissue samples from people who had AD (Vega et al., 2008).

Proteins in the EF Hand family contain the namesake domain, which binds calcium. EFhd2 contains two of these motifs working in tandem, as well as an amino-terminal polyalanine region and a carboxyl-terminal coiled-coil domain. Calcium binding stabilizes EFhd2’s structure, the Vega group has found (Ferrer-Acosta et al., 2012).

In her poster and recent paper, Ferrer-Acosta explored EFhd2’s propensity to aggregate. "So far, this protein seems to have all the characteristics of a new amyloid protein," she said. In brain slices from people who died of AD, EFhd2 appeared in inclusions both on its own and with tau. In vitro, it formed filamentous structures that associated with the amyloid marker thioflavin S. The coiled-coil domain, she found, participated in the EFhd2 oligomerization as well as tau-EFhd2 interactions.

Because intracellular calcium levels may rise during neurodegeneration, the researchers hypothesized that in the case of disease, excess calcium might bind to EFhd2, causing it to aggregate with tau. However, in-vitro experiments contradicted this theory. In fact, the team found that including calcium in the reaction reduced EFhd2’s formation of thioflavin S binding amyloids. Thus, Vega proposed that calcium binding stabilizes EFhd2 in its proper, physiological form. The less stable version without calcium, then, would be the pathogenic one.

Recombinant EF Hand 2 forms filaments in vitro. Image courtesy of Yancy Ferrer-Acosta and François Orange, University of Puerto Rico

What prevents intracellular calcium from interacting with EFhd2 in the disease state? The work presented by Vázquez-Rosa may provide an answer. He reported that, like tau, EFhd2 is a substrate for the Cdk5/p25 kinase complex. Cdk5/p25 attaches a single phosphate to EFhd2, at serine 74, in vitro. The same event occurred in brain extracts from JNPL3 mice. It also interfered with calcium binding, Vega said. Thus, the Cdk5/p25 hyperactivity associated with neurodegeneration might prevent EFhd2 from picking up calcium, pushing the protein away from its stable, physiological shape towards a more unstructured, pathological form likely to aggregate. Vega noted that at this point, the data do not indicate whether EFhd2 amyloids are toxic or protective to cells. The EFhd2 structures might act on their own, or promote tau tangle formation, he speculated.

"I think the EF Hand 2 protein is very interesting and seems to play a role in tau pathology," commented Li-Huei Tsai of MIT, who co-organized the meeting, in an e-mail to Alzforum. "It would be important to know whether tau pathology is affected in EF Hand 2 … loss-of-function cells." Tsai has published extensively on Cdk5/p25 (see ARF related news story on Kim et al., 2008; ARF related news story on Samuels et al., 2007; ARF related news story on Sananbenesi et al., 2007), and has shared mouse brain samples with the Vega group.

Rodríguez-Cruz will address the question of EFhd2 loss of function with her knockout mice. The animals, she hopes, will also offer some clues as to EFhd2’s normal function. The oldest mice are at 32 weeks, and she has not yet observed problems with basic brain structure or behavior. In a cage full of animals, it is impossible to tell which are the knockouts and which are wild-type, she said. The researchers can conclude the protein is not essential.

Rodríguez-Cruz and Vega hope that the animals will start to show pathology as they age to perhaps a year or 18 months. If EFhd2 is normally protective, then the researchers predict the knockouts will age faster, Vega said. They also plan to cross their EFhd2 knockouts with the JNPL3 tau mutant strain to see if loss of EFhd2 enhances or rescues the tau pathology.

To address the role of EFhd2 in human disease, Vega is collecting brain samples representing tauopathies and other forms of neurodegeneration. He is eagerly awaiting production of an antibody specific for phosphorylated EFhd2, to examine the phosphorylation state of the protein in human samples. In addition, Vega hopes to start screening DNA samples from people with AD or other conditions for EFhd2 mutations. A few genetic variants in the gene show up in single nucleotide polymorphism databases, he said, but he does not know if those donors exhibited symptoms of neurodegeneration. In addition, Ferrer-Acosta noted that studies in the AlzGene database have linked the region including EFhd2 to late-onset AD (Hiltunen et al., 2001, and Holmans et al., 2005; see also Myers et al., 2002, and Bertram et al., 2007). "These studies suggest that EF Hand 2 may be a risk factor for the development of AD in humans," she wrote in an e-mail to Alzforum.

EFhd2 has received little attention, but Vega hopes that the new paper will help initiate discussion on this and potentially other amyloids beyond the standard-bearers tau and amyloid-β. "There is still a lot to be learned about how tau participates in Alzheimer’s disease," added Sarah Fontaine of the University of South Florida, who was not involved in the study. "There may be more tau-associated proteins which can contribute to the pathology." New tau partners might make good drug targets, she said.—Amber Dance

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References

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Paper Citations

Vega IE, Traverso EE, Ferrer-Acosta Y, Matos E, Colon M, Gonzalez J, Dickson D, Hutton M, Lewis J, Yen SH. A novel calcium-binding protein is associated with tau proteins in tauopathy. J Neurochem. 2008 Jul;106(1):96-106. PubMed.
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