Stable isotope labeling-mass spectrometry as a new approach to determine remyelination

Remyelination and need to access it: A range of diseases such as Guillain-Barre syndrome, Pelizaeus Merzbacher disease, relapsing-remitting and secondary progressive multiple sclerosis is associated with various degrees of nerve demyelination. These diseases present with various degrees of demyelination and different clinical manifestations. Treatments leading to remyelination are important for the restoration of functionality in these diseases with various clinical outcomes. Obtaining proper remyelination remains one of the current limitations for the treatment of demyelinating conditions. Establishing normal nerve function after axonal regeneration also necessitates proper myelination (Franklin et al., 2020). The process of remyelination is in many aspects very similar to the events that occur during development, with the exception that remyelination often does not reach the same myelin sheath thickness as observed during development. Therefore, understanding how these two processes (remyelination versus normal myelination after regeneration) differ can help develop novel translational applications in the field of regenerative medicine. With the scientific exploration of pharmacological compounds and therapies that can induce remyelination, there has been a surge of drugs with the potential to promote remyelination (Wooliscroft et al., 2019). Several of these are currently under clinical trials and include monoclonal antibody therapies, myelin protein stimulants, and non-selective G protein coupled receptor antagonists to name a few (Wooliscroft et al., 2019). However, the question arises as to whether these compounds are promoting remyelination or preventing demyelination. Thus, creating a need to identify newly synthesized myelin from previously existing myelin. Current strategies to assess remyelination: Remyelination is a natural repair mechanism in which progenitor cells differentiate into oligodendrocytes to myelinate axons that have lost their myelin due to axonal injury. Thus, to assess remyelination, methods must be able to differentiate between myelin that is already present and newly synthesized myelin. There are various methods (ex vivo and in vivo) to detect changes in myelination, each with its level of success and limitations. These include histological methods of Luxol fast blue staining combined with transmission electron microscopy. Electron microscopy provides high structural resolution images which allow the measurement of myelin sheath thickness (g-ratio) and currently remain the most reliable hallmark for assessing myelination. Luxol fast blue staining is a method that can provide a means to measure g-ratios; however, it is limited to measuring large-diameter axons and becomes less reliable when applied to smaller-diameter axons, such as those found in the corpus callosum. Both methods provide microscopic detail of myelin sheath structure; however, they are ex vivo, procedurally lengthy, dependent on the correct performance of intricate steps, and cannot differentiate between myelin states (existing myelin versus newly synthesized myelin) thus limiting their application to assess remyelination. Real-time imaging techniques such as magnetic resonance imaging (MRI), myelin water imaging (MWI), quantitative susceptibility mapping, magnetization transfer imaging, and positron emission tomography provide a fast modality for assessing changes in myelination. MRI is a widely used technique and has been correlated with myelination by using different markers, such as fractional anisotropy, radial/transverse diffusivity, susceptibility (quantitative susceptibility mapping), longitudinal relaxation rate (R1), magnetization transfer ratio, and macromolecular pool. However, there is still debate as to how well each marker captures myelin signal, and how each methodology compares with each other (Lazari and Lipp, 2021). MWI is another MRI method that has shown a strong correlation with myelin histology and the ability to detect areas of demyelination and highlight areas that have improved with treatment (Lee et al., 2021). However, MWI is not able to fully capture myelin concentrations and its application is dependent on technical and physiological limitations, therefore MWI should be carefully considered when assessing myelination patterns. Magnetization transfer imaging utilizes the magnetization transfer ratio which has been shown to correlate with myelin histology as well as R1. However, R1 interferes with the magnetization transfer ratio therefore decoupling both parameters demonstrated to have an improved correlation with myelin content. Positron emission tomography imaging also provides a fast modality for assessing myelin changes in vivo and takes advantage of using radiotracers that bind and localize to the myelin sheath. Thus, it serves as a more direct methodology for assessing myelination in real time. Some of these markers include 18F-florbetaben, 18F-florbetapir, 18F-flutemetamol, 11C-MeDAS, and 11C-PiB which are uptake by the brain and localize to the myelin sheath (Auvity et al., 2020). Due to their longer half-life and readily brain uptake, 18F-florbetapir and 18F-florbetaben are promising markers for assessing myelination. Real-time imaging provides a fast modality technique for assessing myelination when histological options are not possible. This has the advantage of monitoring pre and post treatment states but for the most part, is not a highly accurate method to measure remyelination. Other factors such as lesion size and resolution limit the identification of imaging pattern changes, especially for smaller areas. In animal research remyelination is also limited by the ready availability of instruments with high-resolution imaging quality. Non-invasive electrophysiological measurements such as evoked-potentials and pattern electroretinogram are methods that measure compound action potentials and rely on the conduction speed of action-potentials (You et al., 2015; Heidari et al., 2019). During demyelination, axons lose their electrical insulation and thus have a decrease in the electrical transmission that can be measured as latency. Although it is possible to distinguish between a demyelinated nerve and a myelinated nerve, the true state of this measurement might only reflect nerve functionality as opposed to remyelination. Thus, the agreement between histology and electrophysiology should be considered as corroborative methods for remyelination. However, both methodologies are limited by the inability to distinguish between myelin states, as a previously myelinated nerve would have similar electrical conductivities as a newly myelinated nerve and show similar myelin thickness histologically. Being able to distinguish between previously existing and newly synthesized myelin is a crucial step for the identification of remyelination. Other quantitative methods such as western blot assay and enzyme-linked immunosorbent assay detect myelin proteins thereby assessing myelination. They are based on antibody-protein detections and are at best indirect measurements of remyelination. Furthermore, antibody specificity and efficacy are an inherent limitation to these methods. Antibody efficacy varies greatly from vendor to vendor and even between different lots of antibodies from the same batch due to differences in length of storage and variable transport conditions. However, all these approaches do not unequivocally establish whether remyelination is due to the synthesis of new molecules. Identification of protein isoforms associated with remyelination (western blot assay and enzyme-linked immunosorbent assay) together with structural identifying methods (electron microscopy, histology, electrophysiological, and real-time imaging techniques) provide corroborative evidence in parts, that complement and sum up to provide greater information than that provided by any one of these modalities alone. Incorporation of isobaric C13-amino acids to assess remyelination: Changes in protein synthesis are integral processes in health and disease and contribute to our understanding of the proteome of these two states. Isobaric labels and mass spectrometry may provide unequivocal molecular evidence of the new synthesis of myelin proteins. Thus, providing a direct method for differentiating myelin states and establishing remyelination. There are at least 14 different isoforms of myelin basic protein expressed in the murine central nervous system (Valdivia et al., 2019). These are divided into two families 1) the Golli (Genes of Oligodendrocyte Lineage) family which is expressed ubiquitously throughout the immune and central nervous system and 2) the classic family which is expressed by oligodendrocytes. Within the classic family, myelin basic protein isoform 5 has been implicated in demyelinating conditions and has been promoted as the source of immunogenic peptides that contribute to demyelination (Musse et al., 2006). Therefore, the identification of structural proteins that stabilize the myelin sheath (such as myelin basic protein isoform 5) can be captured by C13 labeled amino acids as it will be incorporated in the peptide sequence when synthesized de novo. The capture of newly synthesized myelin proteins may help distinguish myelin states in demyelinating diseases such as multiple sclerosis and neuromyelitis optica and might even be able to capture new remyelinating events happening within minutes prior to biopsy collection. Several approaches can detect and quantify newly synthesized proteins which include puromycin-based methods, bio-orthogonal noncanonical amino acid tagging, and stable isotope labeled amino acids. However, most of these approaches have largely been limited to their application in cell cultures. In our recent study (Valdivia and Bhattacharya, 2022), we utilized mouse models of multiple sclerosis to assess the remyelinating effects of a lysophospholipid injected directly in the sub-meningeal space of the optic nerve. We observed recovery of optic nerve electrical activity (as measured by pattern electroretinogram), maturation of oligodendrocytes from oligodendrocyte progenitor cells (detected by markers such as platelet-derived growth factor receptor-α) to mature myelinating oligodendrocytes (detected by myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein) and increase myelin sheath thickness (observed by transmission electron microscopy). To access remyelination, we utilized isobaric C13-Histidine in the optic nerve to label and detect de novo myelin protein synthesis (Figure 1; Harvey et al., 2023). Already existing myelin would not incorporate C13-Histidine in myelin proteins such as MBP, whereas newly synthesized myelin would require de novo protein synthesis and thus incorporation of C13-Histidine. Our results demonstrated the incorporation of C13-Histidine at H:22, H:25, H:31, and H:80 of the MBP protein sequence (detected by mass spectrometry spectra shift in m/z, H and number refer to histidine within the canonical MBP isoform 5 protein sequence). These findings were mechanistically further validated by culturing oligodendrocyte progenitor cells in the presence of a lysophospholipid which demonstrated oligodendrocyte maturation (presenting MBP and oligodendrocyte-specific protein markers) thus supporting remyelination induced by this lysolipid when compared to a known demyelinating agent. We used C13-Histidine to establish remyelination in our studies but any other stable isobaric labels (those amino acids that are in the sequence of myelin proteins) are just as good.Figure 1: Incorporation of stable isotope labeled amino acid in the myelin sheath during remyelination.(A) Comparison of different myelin states during health and demyelinating diseases such as multiple sclerosis. (B) Treatment of demyelination by remyelinating agents (neuregulin or lysolipid) along with stable isotope C13-Histidine. (C) Incorporation of stable isotope C13-Histidine in newly synthesized myelin sheath proteins. (D) Identification of incorporated stable isotope C13-Histidine in myelin protein sequence by mass spectrometry. Created with BioRender.com.Limitations of using a stable isotope labeled amino acids approach: The incorporation of isobaric labeled amino acids serves as a reliable approach to quantify and assess remyelination. Using multiple labeled amino acids is likely to provide a more reliable and sensitive method along with the benefit of using lower and safer concentrations of each single labeled amino acids in the mix. This methodology, however, also poses inherit limitations. The induction of remyelination by administering C13-Histidine in vivo along with remyelinating agent(s), will be preceded simultaneously with other molecular events that will require de novo protein synthesis. These preceding events will deplete available C13-Histidine, therefore, reducing the amount available to be incorporated in myelin proteins. The natural solution to this limitation would be to increase C13-labeled amino acids concentrations. However, increasing C13-Histidine concentrations had detrimental effects on optic nerve function as measured by a decrease in pattern electroretinogram amplitude in our recent publication (Valdivia and Bhattacharya, 2022). These detrimental effects are expected to be carried to other applications of this method. However, the use of multiple stable labeled amino acids may partly help overcome this limitation as noted above. Variations between different instruments will require individual optimizations of the method for each instrument, as this will affect the detection and identification of incorporated C13-Histidine or other stable isobaric labeled amino acids. The health effects of long-lasting incorporation of artificial stable isotope labeled amino acids are yet to be fully investigated and thus remain a limitation. Future directions: It will be useful to combine molecular identification with tissue localization. Two advances towards this localized molecular identification show promising applications for the future, these are: (1) emerging imaging mass spectrometry techniques that may help identify the topographical distribution of remyelination in tissue using the incorporation of stable isotope-labeled amino acids. This technique has the additional benefit of using a small sample size and tiny amounts of biopsy tissues. The possibility of using stable isotopes ex vivo on living tissues is a potential future development that will eliminate the consideration of long-term toxicity issues. (2) Emerging mass spectrometry methods to analyze protein-lipid complexes directly from membranes (Chorev et al., 2018) combine top down and bottom-up mass spectrometry approaches. This method opens the possibility for investigating the interaction of myelin proteins with the myelin lipid membrane, thus allowing us to further our understanding of protein-lipid interactions during remyelination. Conclusion: A major advantage of utilizing the incorporation of stable isotope labeled amino acids is the ability to distinguish previously existing myelin versus newly synthesized myelin. This is a crucial distinction that is often a limiting factor to other ex vivo approaches and thus serves as a novel approach for assessing remyelination. Furthermore, this methodology is not only limited to implementation in neuronal tissue but can be applied to various similar biological systems for the determination of newly synthesized proteins. This versatility makes this method suitable to incorporate in experimental designs for the validation of remyelination potential in novel therapeutics. It also offers the opportunity to gain insights into the process of remyelination in demyelinating conditions and opens an avenue for potential applications in translational medicine. This work was supported by the US Department of Defense Grant WH-X81160715; Department of Health and Human Services | National Institutes of Health | National Eye Institute Grants EY-027257 and EY-14801 (to SKB); and an unrestricted grant to the University of Miami from Research to Prevent Blindness. C-Editors: Zhao M, Liu WJ, Qiu Y; T-Editor: Jia Y