ALS Modifier: Ephrin Receptor Loss Delays Onset, Progression

29 August, 2012. Researchers have discovered a gene that adjusts the course of amyotrophic lateral sclerosis, a fatal and rapidly progressing neurodegenerative disease. When the ephrin receptor EphA4 is mutated or missing, it delays disease onset and slows progression in a variety of model organisms. Scientists led by Wim Robberecht of VIB Leuven, Belgium, also found that two people who had ALS plus an EphA4 mutation lived at least seven years after symptom onset—a long time for this quick-moving neurodegenerative disease. The findings were published in the August 26 Nature Medicine online.

“This is a seminal study showing that EphA4 expression in motor neurons is an important factor in animal models of ALS and, critically, that reduced EphA4 expression correlated with less severe disease in humans,” wrote Andrew Boyd of the Queensland Institute of Medical Research in Herston, Australia, in an e-mail to Alzforum (see full comment below). Boyd was not involved in the study.

As a receptor tyrosine kinase, EphA4 mediates signals that repel axons during brain development. It is also expressed in adult brain, where it appears to regulate synaptic architecture and plasticity (for a review, see Klein, 2009). EphA4 modulates the actin cytoskeleton that controls neuronal growth cones (Shi et al., 2007). It is not the first axon- and actin-regulating protein linked to ALS; profilin, a recently discovered ALS gene, catalyzes actin polymerization and growth cone formation (see ARF related news story on Wu et al., 2012).

EphA4 turned up as the strongest hit that Robberecht and first author Annelies Van Hoecke discovered in a screen for genetic modifiers of motor neuron degeneration. They set out to investigate the variability in age of onset of ALS and in its speed of progression. Some people succumb to ALS in their thirties, while others remain healthy into their eighties before exhibiting symptoms; lifespan ranges from a few months to more than 20 years after disease onset. “Even in families with the same mutation, there is a large variability,” Robberecht said (Penco et al., 2011; Kim et al., 2007).

Van Hoecke used a zebrafish model of ALS based on expression of human mutant superoxide dismutase 1 (mSOD1) . Mutant SOD1 shortens and promotes excess branching of motor neuron axons in these animals (Lemmens et al., 2007). Van Hoecke and colleagues screened 303 different antisense oligonucleotides that block translation of their target genes, and came up with five candidates. The fish Rtk2 gene, which corresponds to human EphA4, was the strongest hit. Shutting down Rtk2 or the related Rtk1, via antisense nucleotides or with a chemical that blocks ephrin receptors (Noberini et al., 2008), rescued mSOD1 axonopathy in the fish.

To see if the same approach would work in mammals, Van Hoecke crossed mSOD1 mice with animals missing one copy of the ephrin receptor gene. In the mSOD1/EphA4 heterozygotes, the 50 percent reduction of EphA4 expression prolonged survival by approximately two weeks compared to mSOD1-only control mice, although the age of symptom onset was the same. The EphA4 heterozygote transgenics also possessed more motor neurons and healthier neuromuscular junctions than did their mSOD1 counterparts.

The story was slightly different in rats. A peptide that blocks EphA4 (Fabes et al., 2007) delayed the onset of symptoms in animals expressing human mSOD1, but did not affect progression. That blocking of EphA4 slowed progression in mice, but delayed onset in rats, could be due to differences between the models or the EphA4 blockers used in each case, the authors suggested.

“The bottom line from the fish, mouse, and rat experiments was that the less EphA4 was expressed, the better,” Robberecht said. Human studies told the same story. Van Hoecke and colleagues analyzed EphA4 mRNA levels in blood samples from 158 Dutch people with ALS, and observed that the less EphA4 people had, the later in life they developed the disease, and the longer they survived with it.

What explains the variability in EphA4 levels in the human population? Genomewide association studies did not uncover any variants near EphA4 that associated with disease severity. However, collaborator Robert Brown, at the University of Massachusetts Medical School in Worchester, sequenced EphA4 in people with ALS and came across two new variants: a stop codon in place of arginine 514, and a substitution of glutamine for a conserved arginine at position 571. A man with the truncation lived for seven years after symptoms started, and a man with a missense mutation has survived 12 years and counting, after diagnosis. ALS normally runs its course in two to three years. The data suggest that the receptor makes an attractive target for pharmaceutical intervention, Robberecht said.

Robberecht and colleagues will investigate the mechanism by which EphA4 enhances ALS. The current study hints that neurons with lots of the receptor are prone to degenerate. Van Hoecke used laser-capture microdissection to isolate individual spinal motor neurons from mice and measure EphA4 mRNA. She discovered that the large neurons, which are most vulnerable to ALS, make more EphA4 than the smaller, resistant neurons. The relationship between EphA4 and disease susceptibility may hold true in other neurodegenerative diseases as well, the authors suggest. They found that blocking EphA4 in fish expressing mutant TDP-43, another ALS gene, or the spinal muscular atrophy gene Smn1, also rescued axonal pathology.—Amber Dance

Comments

  1. The study by Van Hoecke et al. not only identifies EphA4 as a potential therapeutic target for amyotrophic lateral sclerosis (ALS), but it also reveals an unexpected role of this receptor tyrosine kinase in determining the disease duration of ALS. The authors show that reduction of EphA4 expression rescues motor axonopathy and significantly prolongs the survival of the SOD1 mutant mice, an ALS mouse model. Importantly, the beneficial effect of inhibiting EphA4 in slowing down the disease progression in these mice is verified by administration of EphA4 inhibitors. Intriguingly, they also demonstrate that the relative EphA4 expression in the blood of ALS patients is inversely correlated with the age of disease onset, and two EphA4 variants are identified in patients associated with uncharacteristically long survival. These observations together unequivocally indicate that EphA4 is a critical determinant of ALS disease progression and open a new avenue in ALS research.

    The study is remarkable in a number of aspects. First, it demonstrates the success of using lower vertebrates, such as zebrafish, as a model system to screen for signaling molecules that are crucial for the progression of neurodegenerative diseases. Second, it identifies EphA4 as the key player in regulating motor neuron survival, thus revealing the therapeutic potential of EphA4 inhibitors in ALS. It would be of interest to examine whether downstream effectors of EphA4 such as α2-chimaerin are involved in the disease as well (Dalva, 2007; Shi et al., 2007). Finally, findings from this study raise the intriguing possibility of using pharmacological inhibitors of EphA4 as a therapeutic strategy for neurodegenerative diseases. Our laboratory and others have demonstrated that EphA4 is a key negative regulator of neurotransmission in the brain by promoting dendritic spine retraction and degradation of neurotransmitter receptors (Murai et al., 2003; Fu et al., 2007; Fu et al., 2011). Since deficits in synaptic transmission and plasticity are associated with neurodegenerative diseases such as Alzheimer’s disease, EphA4 inhibitors might represent promising therapeutic agents for these diseases.

    References:

    Dalva MB. There's more than one way to skin a chimaerin. Neuron. 2007 Sep 6;55(5):681-4. PubMed.

    Fu AK, Hung KW, Fu WY, Shen C, Chen Y, Xia J, Lai KO, Ip NY. APC(Cdh1) mediates EphA4-dependent downregulation of AMPA receptors in homeostatic plasticity. Nat Neurosci. 2011 Feb;14(2):181-9. Epub 2010 Dec 26 PubMed.

    Fu WY, Chen Y, Sahin M, Zhao XS, Shi L, Bikoff JB, Lai KO, Yung WH, Fu AK, Greenberg ME, Ip NY. Cdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanism. Nat Neurosci. 2007 Jan;10(1):67-76. PubMed.

    Murai KK, Nguyen LN, Irie F, Yamaguchi Y, Pasquale EB. Control of hippocampal dendritic spine morphology through ephrin-A3/EphA4 signaling. Nat Neurosci. 2003 Feb;6(2):153-60. PubMed.

    Shi L, Fu WY, Hung KW, Porchetta C, Hall C, Fu AK, Ip NY. Alpha2-chimaerin interacts with EphA4 and regulates EphA4-dependent growth cone collapse. Proc Natl Acad Sci U S A. 2007 Oct 9;104(41):16347-52. PubMed.

    View all comments by Nancy Ip

  2. This is a seminal study showing that EphA4 expression in motor neurons is an important factor in animal models of ALS and, critically, that reduced EphA4 expression correlates with less severe disease in humans.

    When we developed the EphA4-/- mouse, it revealed the crucial role of this protein in motor axons. We thus predicted an important role in neurological diseases affecting motor nerves. In this study by Van Hoecke et al., it was not possible to use the EphA4-/- animals, but the EphA4+/- x SOD1 cross had significantly slowed ALS disease progression. Thus, inhibitors which reduce EphA4 function to less than 50 percent would be predicted to slow disease to at least this extent. We have recently reported a positive therapeutic effect of an EphA4 inhibitor on motor function after spinal cord injury (Goldshmit et al., 2011), and therefore strongly support the suggestion that such agents may have a beneficial role in ALS.
     

    References:

    Goldshmit Y, Spanevello MD, Tajouri S, Li L, Rogers F, Pearse M, Galea M, Bartlett PF, Boyd AW, Turnley AM. EphA4 blockers promote axonal regeneration and functional recovery following spinal cord injury in mice. PLoS One. 2011;6(9):e24636. PubMed.

    View all comments by Andrew Boyd

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References

News Citations

  1. Profilin Gene Is Actin’ in ALS

Paper Citations

  1. Klein R. Bidirectional modulation of synaptic functions by Eph/ephrin signaling. Nat Neurosci. 2009 Jan;12(1):15-20. Epub 2008 Nov 23 PubMed.
  2. Shi L, Fu WY, Hung KW, Porchetta C, Hall C, Fu AK, Ip NY. Alpha2-chimaerin interacts with EphA4 and regulates EphA4-dependent growth cone collapse. Proc Natl Acad Sci U S A. 2007 Oct 9;104(41):16347-52. PubMed.
  3. Wu CH, Fallini C, Ticozzi N, Keagle PJ, Sapp PC, Piotrowska K, Lowe P, Koppers M, McKenna-Yasek D, Baron DM, Kost JE, Gonzalez-Perez P, Fox AD, Adams J, Taroni F, Tiloca C, Leclerc AL, Chafe SC, Mangroo D, Moore MJ, Zitzewitz JA, Xu ZS, van den Berg LH, Glass JD, Siciliano G, Cirulli ET, Goldstein DB, Salachas F, Meininger V, Rossoll W, Ratti A, Gellera C, Bosco DA, Bassell GJ, Silani V, Drory VE, Brown RH, Landers JE. Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis. Nature. 2012 Aug 23;488(7412):499-503. PubMed.
  4. Penco S, Lunetta C, Mosca L, Maestri E, Avemaria F, Tarlarini C, Patrosso MC, Marocchi A, Corbo M. Phenotypic heterogeneity in a SOD1 G93D Italian ALS family: an example of human model to study a complex disease. J Mol Neurosci. 2011 May;44(1):25-30. PubMed.
  5. Kim W, Kim JS, Lee KS, Gwoun YJ, Kim JM, Lee KH. Anticipation and phenotypic heterogeneity in korean familial amyotrophic lateral sclerosis with superoxide dismutase 1 gene mutation. J Clin Neurol. 2007 Mar;3(1):38-44. PubMed.
  6. 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.
  7. Noberini R, Koolpe M, Peddibhotla S, Dahl R, Su Y, Cosford ND, Roth GP, Pasquale EB. Small molecules can selectively inhibit ephrin binding to the EphA4 and EphA2 receptors. J Biol Chem. 2008 Oct 24;283(43):29461-72. PubMed.
  8. Fabes J, Anderson P, Brennan C, Bolsover S. Regeneration-enhancing effects of EphA4 blocking peptide following corticospinal tract injury in adult rat spinal cord. Eur J Neurosci. 2007 Nov;26(9):2496-505. PubMed.

Further Reading

Papers

  1. Goldshmit Y, Spanevello MD, Tajouri S, Li L, Rogers F, Pearse M, Galea M, Bartlett PF, Boyd AW, Turnley AM. EphA4 blockers promote axonal regeneration and functional recovery following spinal cord injury in mice. PLoS One. 2011;6(9):e24636. PubMed.
  2. Wegmeyer H, Egea J, Rabe N, Gezelius H, Filosa A, Enjin A, Varoqueaux F, Deininger K, Schnütgen F, Brose N, Klein R, Kullander K, Betz A. EphA4-dependent axon guidance is mediated by the RacGAP alpha2-chimaerin. Neuron. 2007 Sep 6;55(5):756-67. PubMed.
  3. Fabes J, Anderson P, Yáñez-Muñoz RJ, Thrasher A, Brennan C, Bolsover S. Accumulation of the inhibitory receptor EphA4 may prevent regeneration of corticospinal tract axons following lesion. Eur J Neurosci. 2006 Apr;23(7):1721-30. PubMed.
  4. Goldshmit Y, Galea MP, Wise G, Bartlett PF, Turnley AM. Axonal regeneration and lack of astrocytic gliosis in EphA4-deficient mice. J Neurosci. 2004 Nov 10;24(45):10064-73. PubMed.

Primary Papers

  1. Van Hoecke A, Schoonaert L, Lemmens R, Timmers M, Staats KA, Laird AS, Peeters E, Philips T, Goris A, Dubois B, Andersen PM, Al-Chalabi A, Thijs V, Turnley AM, van Vught PW, Veldink JH, Hardiman O, Van Den Bosch L, Gonzalez-Perez P, Van Damme P, Brown RH, van den Berg LH, Robberecht W. EPHA4 is a disease modifier of amyotrophic lateral sclerosis in animal models and in humans. Nat Med. 2012 Sep;18(9):1418-22. PubMed.

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