12 Nov 2016

In the search for structural biomarkers that track the progression of amyotrophic lateral sclerosis (ALS), scientists led by Sunney Xie and Kevin Eggan at Harvard University may have discovered the earliest yet. As reported in the October 31 Nature Communications, they used a spectroscopic technique called stimulated Raman scattering (SRS) microscopy to follow the breakdown of myelin on degenerating nerve fibers in living mouse models. The myelin becomes damaged weeks before researchers can see any axonal defects, immune system changes, or movement problems. As an added plus, SRS microscopy can be done without harming the nerve tissue, allowing scientists to track changes in animals over time. However, since the technique requires an operation to expose the nerve, it is too invasive in its current form to track decline in people with ALS, although researchers think the surgery requirement may change.

“We were all taken aback when we saw the exquisite detail by which we could observe morphology in living animals and postmortem tissue,” Eggan told Alzforum. “SRS has remarkable resolution.”

In SRS microscopy, two coordinated laser beams are aimed at a sample that absorbs photons and then re-emits them, albeit with slightly different energies depending on the structure of the molecules present. In effect, each molecule creates its own unique light signature or spectrum that can be measured by a detector. Scientists choose wavelengths of laser light that interact best with the chemical bonds in the type of molecule they are imaging—in this case, lipids. That way, the technique can pick up that molecule against a backdrop of others. SRS microscopy requires no labeling or sample processing. It was recently used to visualize myelin degeneration in a mouse model of multiple sclerosis. Since degeneration of myelin occurs in ALS, too, the researchers wanted to explore whether SRS microscopy could be used to study peripheral nerve degeneration in models of this disease (Imitola et al., 2011; Apr 2013 news). 

First authors Feng Tian and Wenlong Yang started by imaging the dissected sciatic nerves from four-, eight-, 12-, and 16-week-old SOD1G93A mice, and from wild-type controls. In the latter, SRS detected neat rows of orderly fibers that were punctuated occasionally by nodes of Ranvier, breaks in the myelin that help propel action potentials down the axon. In the ALS nerves, however, oval structures occasionally interrupted the fibers. These were composed primarily of lipids, and immunohistochemistry revealed that they were covered in myelin basic protein, a major component of the myelin sheath. The authors hypothesized that these ovoids were pieces of the myelinating Schwann cells that accumulated in the nerves when axons degenerated. The authors spotted these structures as early as four weeks, and they accumulated progressively with age. On average, a 0.05 mm² section contained nine, 64, 103, and 189 ovoids at four, eight, 12, and 16 weeks, respectively. They turned up in other mouse models of ALS as well, including SOD1G37R (Boillee et al., 2006), AAV-C9ORF72, and a FUS model (Sharma et al., 2015). 

Tian and colleagues next tried this technique serially in living animals. They anesthetized five-week-old SOD1G93A mice, then made an incision in one of the hind legs to expose the sciatic nerve. Since imaging did not damage the tissue, they could zip up the incision and come back to image the same section of nerve again. As in the dissected nerves, the researchers saw ovoid structures in the intact sciatic nerves in animals as young as five weeks of age. The ovoids accumulated progressively over six more weeks.

The results suggested that this technique captures damage in progress, but could it detect a treatment benefit? Minocycline delays disease progression in SOD1G93A mice, though not in human patients (Zhu et al., 2002; Gordon et al., 2007). The researchers treated five-week-old SOD1G93A mice daily with the drug, then periodically imaged their sciatic nerves. After three weeks, treated animals had half the ovoids of vehicle-treated controls, and one-third less at six weeks. This suggests SRS microscopy can tell whether treatment slows disease progression.

The technique appears to work in postmortem human tissue, as well. The researchers compared ventral and dorsal root samples of four autopsied ALS patients and four age-matched controls. Motor neurons from patients’ ventral roots contained an average of 51 ovoids per 50mm² section; controls had no statistically significant signal. “This shows that the formation of lipid ovoid structures happens in ALS patients and is not just specific to the mouse models,” Eggan said.

Because SRS microscopy does not damage tissue, requires no processing, can be automated, and can be carried out in living animals, it represents a major improvement over current methods to detect ALS damage, Eggan said. It picks up on disease-related demyelination that occurs weeks before mice lose motor function, which is currently used to determine when a candidate treatment should be started and whether it slows progression. SRS microscopy will allow researchers to test candidate drugs in animals earlier in their disease, wrote the authors. Its use in people will depend on miniaturizing the detector so it can be inserted close to the nerve via a needle, they suggested.—Gwyneth Dickey Zakaib

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References

News Citations

1. Oligodendrocyte Support System Fails Early in ALS 5 Apr 2013

Research Models Citations

1. SOD1-G93A (hybrid) (G1H)
2. C9ORF72(AAV)(G4C2)66

Paper Citations

1. Imitola J, Côté D, Rasmussen S, Xie XS, Liu Y, Chitnis T, Sidman RL, Lin CP, Khoury SJ. Multimodal coherent anti-Stokes Raman scattering microscopy reveals microglia-associated myelin and axonal dysfunction in multiple sclerosis-like lesions in mice. J Biomed Opt. 2011 Feb;16(2):021109. 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. Sharma A, Lyashchenko AK, Lu L, Nasrabady SE, Elmaleh M, Mendelsohn M, Nemes A, Tapia JC, Mentis GZ, Shneider NA. ALS-associated mutant FUS induces selective motor neuron degeneration through toxic gain of function. Nat Commun. 2016 Feb 4;7:10465. PubMed.
4. Zhu S, Stavrovskaya IG, Drozda M, Kim BY, Ona V, Li M, Sarang S, Liu AS, Hartley DM, Wu DC, Gullans S, Ferrante RJ, Przedborski S, Kristal BS, Friedlander RM. Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice. Nature. 2002 May 2;417(6884):74-8. PubMed.
5. Gordon PH, Moore DH, Miller RG, Florence JM, Verheijde JL, Doorish C, Hilton JF, Spitalny GM, Macarthur RB, Mitsumoto H, Neville HE, Boylan K, Mozaffar T, Belsh JM, Ravits J, Bedlack RS, Graves MC, McCluskey LF, Barohn RJ, Tandan R, . Efficacy of minocycline in patients with amyotrophic lateral sclerosis: a phase III randomised trial. Lancet Neurol. 2007 Dec;6(12):1045-53. PubMed.

Further Reading

Papers

1. Freudiger CW, Min W, Saar BG, Lu S, Holtom GR, He C, Tsai JC, Kang JX, Xie XS. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science. 2008 Dec 19;322(5909):1857-61. PubMed.
2. Tipping WJ, Lee M, Serrels A, Brunton VG, Hulme AN. Stimulated Raman scattering microscopy: an emerging tool for drug discovery. Chem Soc Rev. 2016 Apr 21;45(8):2075-89. Epub 2016 Feb 3 PubMed.

News

1. ALS-FTD Mouse Model Develops Motor Neuron Disease 11 Nov 2016
2. Fluid NfL Shines, Tau PET Dims, in the Hunt for FTD Biomarkers 29 Sep 2016
3. Stressed Out: RNA-binding Protein Inhabits Granules in ALS, FTD 18 Nov 2015
4. Blood Marker May Predict ALS Progression 4 Jun 2015