09 Jul 2011

Sometimes a fresh pair of eyes can see new pathways to solve a problem. Two June papers related to the pathology and treatment of amyotrophic lateral sclerosis (ALS) come from labs focused not on classical motor neuron experiments, but on the study of neuromuscular junctions and vascular genesis. The studies suggest therapeutic potential for both histone deacetylase (HDAC) inhibitors and angiogenin, a protein involved in the creation of new blood vessels that is also a neuroprotective anti-apoptotic (Galluzi et al., 2009).

Chien-Ping Ko of the University of Southern California in Los Angeles studies neuromuscular junctions (NMJs), but his recent Ph.D. graduate Young-Eun Yoo convinced him to take a look at ALS when she heard there was no treatment for the fatal motor neuron-degenerating condition. She found that the broad, potent HDAC inhibitor trichostatin A boosted the number of NMJs and extended lifespan in ALS model mice, suggesting that researchers might look for more specific HDAC inhibitors that could be therapeutically useful. The work was published online June 25 in Experimental Neurology.

Guo-Fu Hu at Tufts Medical Center in Boston, Massachusetts, studies angiogenin. He became interested in ALS five years ago when another group reported angiogenin mutations in people with the disease (see ARF related news story on Greenway et al., 2006; Greenway et al., 2004). Shuping Li, formerly of Hu’s lab and since moved to Zheijang University in Hangzhou, China, led the current study that lays out the pathway between angiogenin and prevention of apoptosis. They published their work in the Journal of Cell Physiology online June 15.

Hu’s study adds to the evidence that problems in apoptosis contribute to neurodegeneration such as in ALS (see ARF related news story on Pedrini et al., 2010; Reyes et al., 2010), commented Scott Oakes of the University of California in San Francisco, who was not part of the study. “Apoptosis is not just a late event, but it can, in fact, be a driver in neuronal loss,” he said.

Trichostatin A Secures Nerves, Muscles Against Degeneration
HDAC inhibitors have been studied in the context of neurodegenerative disease (reviewed in Chuang et al., 2009), but there has been little attention to their potential in ALS, Yoo said. “This is the first time I have seen it used in an ALS model,” commented Brett Morrison of Johns Hopkins University in Baltimore, Maryland, who was not involved in the study. Similarly, Yoo added, few ALS studies have focused on NMJs. She chose trichostatin A because it has broad-ranging, potent activity, and she figured that would give her the best chance of observing an effect.

Yoo treated ALS model mice overexpressing human mutant superoxide dismutase 1 with the drug. She injected trichostatin, or just the delivery vehicle as a control, interperitoneally five days a week. To mimic when people with the disease would begin treatment, she started at the age of 90 days, when the animals were already symptomatic. Although the drug did not return the mice to wild-type-level health, it improved several pathological signs and symptoms of ALS.

The mice who got the drug were stronger, steadier, and longer lived than the vehicle-only animals. By 120 days from birth, the treated mice gripped objects 50 percent stronger than the controls. At the age of 135 days, trichostatin-treated mice were 60 percent better than the others at balancing on a rotating rod. Finally, the treated mice survived an average of 159 days, 10 days longer than untreated ones—a fairly “typical” improvement for treatments in these mice, Morrison said.

Yoo looked for ALS-specific pathology, such as gliosis and glutamate toxicity. Compared to the delivery-only mice, the treated animals suffered less inflammatory gliosis. Their glia also boasted more of the glutamate transporter GLT-1, which presumably would clear excess glutamate from the extracellular space, limiting its toxicity. Moreover, the treated animals retained 25 percent more motor neurons, in the lumbar spinal cord, than did their untreated counterparts at 120 days.

Axonal die-back is another key feature of ALS, so Yoo counted the myelinated axons innervating the diaphragm and hind limb muscles. At 120 days old, the treated mice possessed 32 percent more axons leading to the diaphragm, and 21 percent more leading to the hind muscles, than the untreated fellows. Similarly, twice as many hind limb NMJs, and 21 percent more in the diaphragm, were innervated in the mice that received trichostatin A. To check for muscle atrophy, Yoo weighed the hind limb muscles of the animals and found the gastrocnemii of treated animals tipped the scales at 41 percent heavier than those of controls.

The effects on muscle could be due to trichostatin A action in the myocytes themselves. Alternatively, Morrison noted, “all of these things...could just be from motor neuron survival.” The study cannot distinguish the two possibilities. Another possible explanation Morrison raised is that trichostatin A could directly inhibit mSOD1 expression, thus alleviating disease. Yoo thinks this is not the case, as she has not observed any significant downregulation of mSOD1 in other experiments.

Trichostatin affects so many HDACs that it is likely to have side effects, but more specific HDAC inhibitors might also be protective, Yoo suggested. “It is something that clearly should be looked at to a greater extent in the future,” Morrison concurred, adding that more information on trichostatin’s protective mechanism would help scientists design feasible drugs.

Angiogenin Pathway to Protection
Another way to protect motor neurons and surrounding tissues might be to keep them from killing themselves. Li and Hu have found that angiogenin prevents apoptosis after they remove serum from P19 mouse embryonal carcinoma cells (Li et al., 2010). It also promotes motor neuron survival in cell culture and mSOD1 mice (Kieran et al., 2008).

In the current work, Li and Hu lay out the biochemical pathway linking angiogenin and apoptosis inducing factor (AIF). AIF is normally located in mitochondria, but when it moves to the nucleus—as it does in ALS model mice (Oh et al., 2006)—it chews up DNA, contributing to apoptosis. In addition to angiogenin and AIF, the players include the apoptosis dampener Bcl-2 and the pro-apoptosis proteins caspase-3 and serum polymerase-1 (PARP-1).

The researchers delineate a pathway in which apoptosis normally proceeds from activation of caspase-3, to cleavage of PARP-1, to nuclear translocation of AIF. Angiogenin alters the situation by upregulating Bcl-2, which inhibits caspase-3 and thus the rest of the downstream pathway. Much of this pathway was already known, noted Piera Pasinelli of Thomas Jefferson University in Philadelphia, Pennsylvania, but “they really teased out the mechanism by which angiogenin can be anti-apoptotic…in a Bcl-2-dependent manner,” she said.

To study apoptosis, the researchers starved P19 cells of serum. This treatment normally causes PARP-1 cleavage within a few hours and nuclear translocation of AIF by 24. But with angiogenin in the culture media, PARP-1 stayed whole and AIF remained mitochondrial, confirming that angiogenin blocks apoptosis via this pathway. In a previous study, the authors confirmed that angiogenin also prevents activation of caspase-3 (Li et al., 2010).

The researchers already knew Bcl-2 was upregulated by angiogenin (Li et al., 2010), and suspected it would be crucial to angiogenin’s effects. If so, then removing Bcl-2 should allow apoptosis to proceed unhindered, even in the presence of angiogenin. Li used RNA interference to knock down Bcl-2. In the knocked down, serum-starved cell cultures, angiogenin was less effective at preventing caspase-3 activation, PARP-1 cleavage and AIF translocation.

These data suggest that angiogenin treatment could protect motor neurons in people with ALS. The Hu group, in unpublished experiments, has treated mSOD1 mice with angiogenin; it was “very effective,” he told ARF, with no adverse effects. Now, he plans to go back to those mice and discover whether the protection involves the same pathway he observed in the cell culture. In addition, he is interested in investigating angiogenin’s neuroprotective effects in other conditions such as Alzheimer’s disease.—Amber Dance

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References

News Citations

1. ALS—Study Strengthens VEGF Connection, Potential Biomarkers Proffered 3 Mar 2006
2. In ALS, “Good” Mitochondrial Protein Turns Partner in Crime 18 Jun 2010

Paper Citations

1. Galluzzi L, Morselli E, Kepp O, Kroemer G. Targeting post-mitochondrial effectors of apoptosis for neuroprotection. Biochim Biophys Acta. 2009 May;1787(5):402-13. PubMed.
2. Greenway MJ, Andersen PM, Russ C, Ennis S, Cashman S, Donaghy C, Patterson V, Swingler R, Kieran D, Prehn J, Morrison KE, Green A, Acharya KR, Brown RH, Hardiman O. ANG mutations segregate with familial and 'sporadic' amyotrophic lateral sclerosis. Nat Genet. 2006 Apr;38(4):411-3. PubMed.
3. Greenway MJ, Alexander MD, Ennis S, Traynor BJ, Corr B, Frost E, Green A, Hardiman O. A novel candidate region for ALS on chromosome 14q11.2. Neurology. 2004 Nov 23;63(10):1936-8. PubMed.
4. Pedrini S, Sau D, Guareschi S, Bogush M, Brown RH, Naniche N, Kia A, Trotti D, Pasinelli P. ALS-linked mutant SOD1 damages mitochondria by promoting conformational changes in Bcl-2. Hum Mol Genet. 2010 Aug 1;19(15):2974-86. PubMed.
5. Reyes NA, Fisher JK, Austgen K, VandenBerg S, Huang EJ, Oakes SA. Blocking the mitochondrial apoptotic pathway preserves motor neuron viability and function in a mouse model of amyotrophic lateral sclerosis. J Clin Invest. 2010 Oct 1;120(10):3673-9. PubMed.
6. Chuang DM, Leng Y, Marinova Z, Kim HJ, Chiu CT. Multiple roles of HDAC inhibition in neurodegenerative conditions. Trends Neurosci. 2009 Nov;32(11):591-601. PubMed.
8. Kieran D, Sebastia J, Greenway MJ, King MA, Connaughton D, Concannon CG, Fenner B, Hardiman O, Prehn JH. Control of motoneuron survival by angiogenin. J Neurosci. 2008 Dec 24;28(52):14056-61. PubMed.
9. Oh YK, Shin KS, Kang SJ. AIF translocates to the nucleus in the spinal motor neurons in a mouse model of ALS. Neurosci Lett. 2006 Oct 9;406(3):205-10. PubMed.

Further Reading

Papers

1. Clement AM, Nguyen MD, Roberts EA, Garcia ML, Boillée S, Rule M, McMahon AP, Doucette W, Siwek D, Ferrante RJ, Brown RH, Julien JP, Goldstein LS, Cleveland DW. Wild-type nonneuronal cells extend survival of SOD1 mutant motor neurons in ALS mice. Science. 2003 Oct 3;302(5642):113-7. PubMed.
2. Boillée S, Yamanaka K, Lobsiger CS, Copeland NG, Jenkins NA, Kassiotis G, Kollias G, Cleveland DW. Onset and progression in inherited ALS determined by motor neurons and microglia. Science. 2006 Jun 2;312(5778):1389-92. PubMed.
3. Wu X, Chen PS, Dallas S, Wilson B, Block ML, Wang CC, Kinyamu H, Lu N, Gao X, Leng Y, Chuang DM, Zhang W, Lu RB, Hong JS. Histone deacetylase inhibitors up-regulate astrocyte GDNF and BDNF gene transcription and protect dopaminergic neurons. Int J Neuropsychopharmacol. 2008 Dec;11(8):1123-34. PubMed.
4. Camelo S, Iglesias AH, Hwang D, Due B, Ryu H, Smith K, Gray SG, Imitola J, Duran G, Assaf B, Langley B, Khoury SJ, Stephanopoulos G, De Girolami U, Ratan RR, Ferrante RJ, Dangond F. Transcriptional therapy with the histone deacetylase inhibitor trichostatin A ameliorates experimental autoimmune encephalomyelitis. J Neuroimmunol. 2005 Jul;164(1-2):10-21. PubMed.
5. Leng Y, Chuang DM. Endogenous alpha-synuclein is induced by valproic acid through histone deacetylase inhibition and participates in neuroprotection against glutamate-induced excitotoxicity. J Neurosci. 2006 Jul 12;26(28):7502-12. PubMed.
6. Yamanaka K, Chun SJ, Boillee S, Fujimori-Tonou N, Yamashita H, Gutmann DH, Takahashi R, Misawa H, Cleveland DW. Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis. Nat Neurosci. 2008 Mar;11(3):251-3. PubMed.
7. Ilieva H, Polymenidou M, Cleveland DW. Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond. J Cell Biol. 2009 Dec 14;187(6):761-72. PubMed.

News

1. ALS—Is It the Neurons or the Glia? 3 Oct 2003
2. ALS—Study Strengthens VEGF Connection, Potential Biomarkers Proffered 3 Mar 2006
3. In ALS, “Good” Mitochondrial Protein Turns Partner in Crime 18 Jun 2010
4. Capillary Clearing—Novel Mechanism a Link to Vascular Dementia? 8 Jul 2010
5. Histone Acetylation: Epigenetic Achilles’ Heel of Memory in Aging 6 May 2010
6. Histone Methyls Solidify Memories 15 Mar 2010
7. It’s an HDAC2 Wrap— Memory-suppressing DNA Modifier Identified 12 May 2009
8. DC: Developing But Debatable—Deacetylase Inhibitors for CNS Disease? 11 Dec 2008
9. Trimming Treatment Time Checks Toxicity of Histone Deacetylase Inhibitors 4 Jan 2008