**5. Conclusion and Future Perspectives**

Redox biomarkers are classified by original cellular components and enzyme mechanisms of action in redox homeostasis. Redox status can be assessed by the measurement of reactive chemical species, oxidative or antioxidative enzyme activity, and degradation products derived from proteins, amino acids, lipid membrane, and nucleic acids. Measurement of reactive chemical species and oxidative enzyme activities assesses intensity of oxidative stress, while measurement of antioxidative activity analyses compensatory capacity. Fine measurement of the various redox components may reveal diagnostic, prognostic, or predicative value to differentiate the disease status and progression (Figure 3).

The levels of nitric oxide (NO) metabolites, *S*-nitrosothiol, and the activities of oxidative enzymes including SOD, MPO, and *i*NOS have been found significantly different to patients with MS, compared to healthy controls. The levels of antioxidants including uric acid and selenium, activities of antioxidative enzymes including GSR, catalase, and TRX-PRDX, and concentrations of transcriptional factors Nrf2, PPARs, and PGC-1α have been found significantly changed in MS patients. The protein degradation products including protein carbonyls, 3-NO-Tyr, glutathionylation, AOPPs, and AGEs, an amino acid by-product ADMA, the lipid and cholesterol degradation products including F2-isoP, MDA, 4-HNE, HODE, oxocholesterols, and oxLDL, and the nucleic acid degradation product 8-OH2dG significantly increased in samples of MS patients. Thus, the oxidative enzymes, antioxidative enzymes, and redox degradation products have been identified as promising biomarkers for the diagnosis of MS. Furthermore, SOD, 3-NO-Tyr, and MDA are sensitive to subtypes of MS, CIS and RRMS, RRMS and SPMS, RRMS and remission, respectively. tNOx, *S*-nitrosothiol, SOD, MPO, GSR, catalase, protein carbonyls, AOPPs, F2-isoP, MDA, and oxycholesterols were correlated with EDSS and thus they are potential prognostic biomarkers for MS. SOD, Nrf2, protein carbonyls, 3-NO-Tyr, AOPPs, and MDA were observed sensitive to the treatment of MS, being possible predictive biomarkers. Finally, SOD is a possible drug target of MS as a therapeutic marker (Table 3).

**Figure 3.** Classification of redox biomarkers according to cellular structures, biomarkers, and their modes of action in redox homeostasis.

In addition to biomarkers described above, the search for novel biomarkers has become a growing interest in neurodegenerative diseases. A micro RNA (miRNA) is a short non-coding RNA molecule consisting of approximately 22 nucleotides, which functions in posttranscriptional gene silencing. miRNAs have been linked to pathogenesis of various diseases including cancer, autoimmune diseases, and neurodegenerative disease such as PD [226]. Dysregulated interactions of miRNAs have been reported in mild cognitive impairment and AD. The associated genes were related to regulation of ageing and mitochondria [227]. Dysregulations of various miRNAs have been observed in AD, PD, Huntington's disease, and ALS and thus miRNAs were proposed to be potential diagnostic and therapeutic biomarkers of neurodegenerative diseases [228]. The link between environmental factors and miRNA dysregulations in MS was discussed [229]. Furthermore, miRNAs in blood and CSF samples of patients with MS as diagnostic and prognostic biomarkers have been reviewed recently [230].

Long Interspersed Nuclear Element-1 (LINE-1) is an autonomous non-long terminal repeat retrotransposon that creates genomic insertions through an RNA intermediate. The increased number of germline and somatic LINE-1s have been linked to the risk and progression of cancer as well as neurodegenerative and psychiatric diseases. An increased burden of highly active retrotranposition competent LINE-1s have been associated with the risk and progression of PD and LINE-1s were proposed as possible therapeutic biomarkers that can be targeted by reverse transcriptase [231]. Furthermore, LINE-1s was considered involved in irregular immune response and participate in pathogenesis of MS [232].

A search for demographic correlation between single nucleotide polymorphisms and MS has been under extensive study. Inflammation-mediating chemokine receptor V Δ32 deletion was not found correlated with MS [233]. Large-scale genome projects such as genome-wide association (GWA) studies generated polygenic risk scores for prediction of risk and progression of multifactorial neurodegenerative diseases. Large-scale pathway specific-genetic risk profiling expedited redox-related biological pathways to identify causal genes and potential therapeutic targets [234]. One of future challenges is a search for correlations with uncatalogued structural variants in MS.

Considering the dynamics of redox homeostasis, the amounts of reactive species, activities of oxidative and antioxidative enzymes, and concentrations of degradation products presumably differ during the progression of MS. The early phase presents an elevation of oxidative enzyme activity and a subsequent elevation of the activity of counteracting antioxidative enzymes with unchanged levels of degradation products. As the activity of antioxidative enzymes becomes compromised due to increasing oxidative stress, the amount of degradation products gradually increases, while the antioxidative enzyme activities slowly wane and fatigue. Eventually, the antioxidative response exhausts with elevated activities of oxidative enzymes and elevated levels of degradation products (Figure 4). Further studies and exploration into novel biomarkers are expected in search of a robust battery of biomarkers indicative to the redox status, in order to realize a fine calibration of major redox components that helps identify the disturbance of redox homeostasis, restore the nucleophilic tone and the most importantly build the best personalized treatment of MS for the sake of better quality of life [235].

**Figure 4.** Dynamics of redox components in disease progression. (**A**) **–** (red line): reactive chemical species and activities of oxidative enzymes increase gradually. **–** (orange line): the degradation products increase accordingly upon exhaustion of antioxidative enzymes' activities. **–** (green line): antioxidative enzymes offset the effects of oxidative enzymes in the early phase, however; the antioxidative activities decline and, finally, fatigue in the later phase, resulting in the exacerbation of inflammation and cellular damage. (**B**) Biomarkers of three redox components may present different values. (a) The levels of oxidative enzyme slightly increase, but the degradation markers stay in a normal range. The antioxidative markers also slightly increase. (b) The oxidative markers further elevate, and the degradation markers start slightly increasing. The antioxidative markers also stay elevated. (c) The oxidative enzyme markers increase, but antioxidative markers return to normal range. The degradation product markers further elevate. (d) The oxidative markers greatly increase, but the antioxidative markers decline. The degradation products markers greatly elevate. : oxidative biomarkers; : degradation biomarkers; : antioxidative biomarkers; ↑: increase; ↓: decrease; and 0: unchanged.

**Author Contributions:** Conceptualization, M.T. and L.V.; writing—original draft preparation, M.T.; writing—review and editing, M.T., and L.V.; visualization, M.T.; project administration, M.T.; supervision, L.V.; funding acquisition, L.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** The current work was supported by GINOP 2.3.2-15-2016-00034, Stay Alive GINOP 2.3.2-15-2016-00048, TUDFO/47138-1/2019-ITM University of Szeged Open Access Fund (FundRef), Grant number 4829.

**Conflicts of Interest:** The authors declare no conflict of interest.
