First-Attack Multiple Sclerosis Targets Gray Matter

Using a highly sensitive and precise proteomics technique, researchers have discovered a handful of biomarkers in cerebrospinal fluid (CSF) that distinguish a first attack of multiple sclerosis (clinically isolated syndrome, CIS) from relapsing-remitting multiple sclerosis (RRMS), as well as from controls.

Particularly notable is the fact that the differences in the CSF proteomes that distinguished first-attack CIS from RRMS and controls occurred in the levels of gray matter-related components, not in myelin markers, according to lead investigator Steven E. Schutzer, M.D., a professor of medicine at Rutgers University New Jersey Medical School in Newark, New Jersey. His findings are published in PLoS ONE (Schutzer et al., 2013).

The investigators also compared first-attack CIS samples to published protein lists derived from CSF samples from healthy normal controls and from CSF from patients with other neurologic diseases with confounding symptoms, namely chronic fatigue syndrome and neurologic post-treatment Lyme disease syndrome.

Fine discrimination of the protein profiles among these groups depended on enabling technology involving high-resolution two-dimensional liquid chromatography coupled to high-performance tandem mass spectrometry (2D-LC-MS/MS) developed over the past decade at the Pacific Northwest National Laboratory in Richland, Washington. The combination of protein preparation with 2D-LC-MS/MS allowed the proteins to be identified. The samples were subjected to immunoaffinity depletion of the most abundant (and therefore masking) proteins that make up the bulk of the protein mass in CSF.

These techniques can now “identify more proteins and more accurately with higher confidence” to see if particular protein signatures are markers for specific patient groups or disease states, Schutzer told MSDF.

Using pooled or at times individual samples from first-attack CIS patients who eventually met the criteria for a diagnosis of MS (n = 9) or RRMS (n = 12) or controls (n = 6), Schutzer and colleagues identified thousands of proteins in CSF, of which 1337 were unique to MS CSF. They performed lumbar punctures within 8 weeks of symptom onset for the CIS patients or relapse for seven of the RRMS patients and for all of the patients with symptoms of other central nervous system disease.

Turning the focus to gray matter

Among the thousands of proteins identified, 20 distinguished first-attack CIS from RRMS and control CSF. Nine were significantly increased, and five were significantly decreased. The remaining six were increased in first-attack CIS compared with RRMS but decreased in relation to control CSF (all P < 0.05).

In characterizing the 20 proteins, “it became apparent that most of those proteins were gray matter-related proteins, not white matter,” Schutzer said. At least 15 of them affected the function of synapse, axon, or neuron. The most striking increase was an 8.04-fold increase in Nogo receptor in the first-attack spinal fluid samples versus RRMS samples and a 2.62-fold increase compared with controls. Nogo receptor regulates axonal growth, regeneration, and synaptic recovery and decreases levels of amyloid beta.

Among the five gray matter proteins with decreased abundance in first-attack CIS versus RRMS CSF, the greatest decreases were in neogenin (-2.83-fold) and chitinase-3-like 1 protein (-2.81-fold). Myelin proteins were detected in both the first-attack CIS and the RRMS samples but did not differ in abundance between the two.

“This is where it became apparent that most of those proteins were gray matter-related proteins, not white matter,” Schutzer said. This finding complements accumulating evidence from very high-resolution research magnetic resonance imaging of gray matter involvement. These gray matter-related proteins at this point may be indicative of an immune attack on the gray matter but may not necessarily be the specific targets. “We don't know exactly what's attacked,” Schutzer says. “We know what results.”

“A completely novel concept”

Senior author Patricia K. Coyle, M.D., professor and vice chair of neurology and director of the Multiple Sclerosis Comprehensive Care Center at Stony Brook University in Stony Brook, New York, added, “You might think of it as the injury marker or the injury signature that's being detected in the spinal fluid.”

She says the results of this research may give insight into the disease process as well as clues to more effective therapy. CIS patients and RRMS patients are two groups at different points along the pathway of the same disease and are distinguishable by differences in gray matter components. “To me it suggests that the first attack of MS, the first relapse of MS, is not as previously believed simply chance happenstance in hitting an eloquent area but rather is orchestrated, that there has to be dysfunction of perhaps critical gray matter components that lead to the first relapse,” Coyle told MSDF. “And that's a completely novel concept in MS.”

She says the data also therefore suggest, but do not prove, that gray matter involvement may be a necessary initiating event. “I think it's telling us something important about why that first attack occurred. It may be important insight into the disease,” she said.

Schutzer emphasized that the current work is at the level of discovery. The next steps are verification of the findings and validation. Verification is done on single samples with the hope that at least 80% will have distinguishing biomarkers in common. If the work is validated, a more commonly available sort of test for the biomarkers, such as an immunoassay, is “very likely,” he said.

Earlier, more targeted therapies?

Coyle foresees such a test being used widely because “at the current time there is no single diagnostic test for MS.” Data indicate that early treatment is key to preventing disability, and a test for very early disease may allow earlier intervention. She even postulated that if caught early enough, “induction strategies that could freeze MS” may be a possibility.

But in the near term, the identification of critical gray matter targets that allow the disease to present could lead to a more antigen-specific approach, possibly blocking the immune attack against those targets.

Asked for an independent perspective on the research, Laura Banks, M.D., medical director of the Monterey Neurological Institute in Monterey and affiliated with the Natividad Medical Center and Community Hospital in Salinas, California, told MSDF, “I think it is an excellent first start. … It corroborates that there is early cortical involvement seen on images from 7-tesla MRI. … To have more physiological data is very helpful.”

Banks said if the findings are validated, it may be possible to select patients for disease-modifying drugs. “I see a huge group of people who don't want to be on disease-modifying treatment, and if I could tell them there's already cortical involvement, that might trigger them to take treatments more seriously,” she said. She also expressed hope that someday proteomic profiles could lead to better prescribing of current drugs.

Additionally, the technology and findings, if validated, could have more immediate diagnostic utility. “There are 5% to 10% of MS patients that don't have distinct lesions on MRI, and spinal taps with oligoclonal bands and IgG synthetic rates are only positive about 50% of the time,” she noted. On the other hand, for many people with migraines with auras and “a couple of spots on MRI,” a clinician may call it MS. “It would be very helpful, especially in an ELISA form, if we could easily rule out people who aren't going to develop the disease and don't have to start the disease-modifying therapy,” obviating daily injections, adverse effects, and attendant costs, Banks said.

A main limitation of the work, she said, was the small number of CSF samples tested. One solution she suggested was to turn to a brain bank run by Claudia Lucchinetti, M.D., at the Mayo Clinic College of Medicine in Rochester, Minnesota. “I think they also have stored CSF samples, and she separated them by histopathology,” so a study on these specimens and samples would be “very, very interesting … if they could find protein signatures for the different pathologies that were found on autopsy.” It would also expand the sample size beyond that in the present study.

Key open questions

• Could this proteomic technique allow the specific diagnosis of RRMS upon first attack?
• Will this proteomic technique allow earlier intervention and the potential for altering the course of the disease?
• Are these gray matter proteins merely biomarkers of an attack on gray matter, or are they in themselves targets of the attack? What is the target within gray matter that allows the clinical presentation of relapsing MS, and could it be used to limit the course of the disease?

Disclosures

Dr. Banks reported no disclosures. Dr. Coyle reported no relationships that could pose the appearance of a conflict of interest regarding the subject of this research. Dr. Schutzer reported no relevant disclosures.