Review

Neurodegenerative Biomarkers in Multiple Sclerosis: At the

Interface Between Research and Clinical Practice
Alin Ciubotaru 1 , Mădălina Irina Smihor 2 , Cristina Grosu 1 , Daniel Alexa 1 , Roxana Covali 3, * ,
Robert-Constantin Anicăi 2 , Ioana Păvăleanu 4 , Andrei Ionut, Cucu 5 , Amelian Mădălin Bobu 6 ,
Cristina Mihaela Ghiciuc 7,8 and Emilian Bogdan Ignat 1

1 Department of Neurology, “Grigore T. Popa” University of Medicine and Pharmacy, 700115 Iasi, Romania;
[email protected] (A.C.); [email protected] (C.G.); [email protected] (D.A.);
[email protected] (E.B.I.)
2 Grigore T. Popa, University of Medicine and Pharmacy, 700115 Iasi, Romania;
[email protected] (M.I.S.); [email protected] (R.-C.A.)
3 Department of Radiology, Biomedical Engineering Faculty, “Grigore T. Popa” University of Medicine and
Pharmacy, 700115 Iasi, Romania
4 Mother and Child Department, “Grigore T. Popa” University of Medicine and Pharmacy,
700115 Iasi, Romania; [email protected]
5 Faculty of Medicine and Biological Sciences, University Stefan cel Mare of Suceava, 720229 Suceava, Romania;
[email protected]
6 Department of Cardiology, Sf. Spiridon Hospital, 700111 Ias, i, Romania; [email protected]
7 Department of Morpho-Functional Sciences II—Pharmacology and Clinical Pharmacology, “Grigore T. Popa”
University of Medicine and Pharmacy, 700115 Iasi, Romania; [email protected]
8 “Saint Mary” Emergency Children Hospital, 700887 Iasi, Romania
* Correspondence: [email protected]

Abstract: Multiple sclerosis (MS) is a chronic autoimmune disorder characterized by in-

flammation, demyelination, and neurodegeneration within the central nervous system
(CNS). While the inflammatory components of MS have been extensively studied, the
progressive neurodegenerative aspect remains a critical factor contributing to long-term
disability. Therefore, the identification and validation of biomarkers associated with neu-
Academic Editors: Giuseppe Lippi
rodegenerative processes are essential for improved diagnosis, prognosis, and treatment
and Camilla Mattiuzzi
monitoring. This review explores cerebrospinal fluid (CSF) and blood-based biomarkers,
Received: 24 March 2025
including neurofilaments, lipid markers, kynurenines, and other molecular indicators that
Revised: 28 April 2025
Accepted: 29 April 2025
provide insights into neurodegeneration in MS.
Published: 6 May 2025
Keywords: multiple sclerosis; neurofilaments; neurodegeneration; kynurenines
Citation: Ciubotaru, A.; Smihor, M.I.;
Grosu, C.; Alexa, D.; Covali, R.;
Anicăi, R.-C.; Păvăleanu, I.; Cucu, A.I.;
Bobu, A.M.; Ghiciuc, C.M.; et al.
Neurodegenerative Biomarkers in 1. Introduction
Multiple Sclerosis: At the Interface
Multiple sclerosis (MS) is a multifaceted disorder that affects both white and gray
Between Research and Clinical
matter structures in the central nervous system (CNS). Traditionally regarded as an in-
Practice. Diagnostics 2025, 15, 1178.
https://doi.org/10.3390/
flammatory demyelinating disease, recent studies have underscored the importance of
diagnostics15091178 neurodegeneration in MS pathology. In contrast to relapsing–remitting MS (RRMS), where
inflammatory processes dominate, the progressive forms of MS show the predominance of
Copyright: © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
neurodegeneration processes. The development of biomarkers capable of distinguishing
This article is an open access article between inflammatory and neurodegenerative changes during the evolution of the disease
distributed under the terms and are crucial for the optimization of therapeutic strategies.
conditions of the Creative Commons The number of people affected worldwide is estimated at around 2.8 million with the
Attribution (CC BY) license
highest prevalence observed in Europe. Multiple sclerosis is the most common chronic
(https://creativecommons.org/
neurological disease among young adults, predominantly affecting women, with a mean
licenses/by/4.0/).

Diagnostics 2025, 15, 1178 https://doi.org/10.3390/diagnostics15091178

Diagnostics 2025, 15, 1178 2 of 18

age of onset of 30 years. The economic and social costs associated with MS, including
loss of productivity and long-term healthcare needs, highlight the importance of early
detection and intervention. A comprehensive understanding of the neurodegenerative
components of MS is crucial for the development of targeted therapeutic interventions
aimed at decelerating that can slow disease progression. Moreover, the heterogeneity of
MS presents challenges in predicting individual disease courses, reinforcing the need for
comprehensive biomarker profiling to personalize treatment strategies effectively.
In recent years, a considerable body of literature has focused on the identification and
validation of neurodegenerative biomarkers in multiple sclerosis (MS). While numerous
reviews have discussed the potential utility of various biomarkers, few have systematically
synthesized quantitative data regarding their diagnostic performance. The present review
aims to address this gap by providing a comprehensive, cohort-specific synthesis of sensitiv-
ity and specificity values for key emerging biomarkers, including neurofilament light chain
(NfL), glial fibrillary acidic protein (GFAP), total tau (t-tau), and others. By integrating de-
tailed cohort characteristics and critically highlighting areas where data remain insufficient,
this work not only offers a practical guide for clinicians but also delineates directions for
future research. Furthermore, by contextualizing these findings within clinical practice, the
present article seeks to enhance the interpretability and applicability of biomarker data.

1.1. Risk Factors of Progression to Secondary Progressive Multiple Sclerosis

There are multiple clinical and demographic risk factors for the progression from
relapse-remitting multiple sclerosis (RRMS) to secondary progressive multiple sclerosis
(SPMS). Older age at disease onset is correlated with an increased risk of conversion.
Furthermore, higher Expanded Disability Status Scale (EDSS) scores at onset and the
presence of spinal cord lesions and cerebellar dysfunction at diagnosis are correlated with
an increased risk of disease progression. Smoking was also shown to be a risk factor for
transitioning to SPMS [1].
From a neurobiological perspective, neuronal exhaustion plays a role in the disease’s
advancement, highlighted by higher neurofilament light chain levels and accelerated gray
matter atrophy. Furthermore, patients who experience high relapse rates in the early stages
of the disease are at greater risk of progressing to SPMS [2].

1.2. Objective of This Review

The objective of this review is to summarize and collect actual data about the present
and possible future neurodegenerative biomarkers for either diagnostic or progression
monitoring used in MS to offer a better understanding of the subject. We will evaluate
the neurodegenerative biomarkers found in CSF, blood, and their correlation with the
clinical evaluation of diagnosis and progression measured by disability. It is important
to note that, in addition to the inflammatory biomarkers, there are multiple promising
biomarkers displaying neurodegeneration in multiple sclerosis (MS). However, these will
not be addressed in this review.
The objective of this study is to elucidate the function of each neurodegenerative
biomarker that has been examined and its potential application in conjunction with MRI
and clinical assessments of disease progression as a decision-support tool for managing
patients with MS [3].
Using multiple biomarkers may prove to be useful in developing a more comprehen-
sive panel that addresses the limitations of using a single biomarker. Consequently, more
research with recent technological and statistical approaches is needed to identify novel
and effective diagnostic and prognostic biomarker tools in MS [4].
Diagnostics 2025, 15, 1178 3 of 18

1.3. Materials and Methods

We conducted a comprehensive literature review focused on neurodegenerative
biomarkers in MS. The search strategy included querying PubMed and relevant databases
for clinical trials, cohort studies, and meta-analyses published in the last two decades that
evaluated biomarkers of neuronal or axonal injury in MS. Keywords used in the search
encompassed terms such as “multiple sclerosis biomarkers”, “neurodegeneration”, “axonal
damage markers”, and specific biomarker names (e.g., “neurofilament”, “tau protein”,
“NfL”, “GFAP”, “YKL-40”, “chitinase”, “NSE”, “14-3-3”, “kynurenine”, “myelin basic pro-
tein”, “extracellular vesicles”, “metabolomics”, and “lipid biomarkers”). Both cerebrospinal
fluid and blood-based biomarkers were included. We also included relevant systematic
reviews and meta-analyses to ensure comprehensive coverage of the topic. The inclusion
criteria for studies were as follows: (1) studies involving patients with MS (any subtype)
that measured one or more of the candidate neurodegenerative biomarkers; (2) reporting
on sensitivity, specificity, or clinical correlations (e.g., with disease activity, progression,
or outcomes); and (3) published in peer-reviewed journals. Data were extracted on study
design, sample size, MS phenotype, the biomarker levels in MS vs. controls, and any
measures of diagnostic performance (sensitivity, specificity) or prognostic significance.
Given the heterogeneity of methods, a qualitative synthesis was performed rather than a
formal meta-analysis (except where meta-analytic data were available from the literature).
The findings for each biomarker are summarized in the Results Section.

1.4. Glossary of Terms

• Diagnostic biomarker: A biomarker used to indicate or confirm the presence of dis-
ease (MS), aiding in the diagnosis (e.g., distinguishing MS from other conditions at
disease onset).
• Prognostic biomarker: A biomarker that predicts the future disease course or outcome
(for example, risk of relapses, rate of disability progression, or conversion to SPMS).
• Monitoring biomarker: A biomarker that reflects current disease activity or treatment
response, used for ongoing tracking of MS and to inform adjustments in therapy.

2. Key Biomarkers in MS and Their Clinical Significance

There are a variety of biomarkers currently under investigation for their potential
to track neurodegeneration, predict disease progression, and assess treatment response
in MS. We will discuss each biomarker in detail, outlining its role and clinical relevance.
According to the World Health Organization (WHO), a biomarker is defined as “any
substance, structure, or process that can be measured in the body or its products and
influence or predict the incidence of outcome or disease”. The characteristics of a good
biomarker need to fit certain criteria: it must be easily reproducible and inexpensive; it
must have good specificity and sensitivity; it must have the ability to produce reliable
results; and it must be obtained through minimal/not-invasive ways [5].
Current Biomarkers:

2.1. Biomarkers of Axonal Damage

2.1.1. Neurofilament Light Chain (NfL)
Neurofilament light chain (NfL) is a well-established biomarker of axonal damage
and neurodegeneration. NfL is released into the cerebrospinal fluid (CSF) and blood
following neuronal injury, making it a reliable indicator of disease activity. Elevated NfL
levels correlate with brain atrophy, disability progression, and response to treatment [6].
As a result, NfL is increasingly used to monitor the effectiveness of disease-modifying
therapies (DMTs) in MS patients [7]. High baseline sNfL levels have been linked to a
Diagnostics 2025, 15, 1178 4 of 18

higher risk of future relapses and new MRI lesions [5]. In other words, an MS patient
with very elevated sNfL levels at onset is likely experiencing subclinical disease activity,
which may signal suboptimal disease control if not addressed. One of the limitations of the
serum NfL is that it is specific for pathology (i.e., neuronal damage) but not for a specific
disease. Elevated levels have been reported in acute conditions such as traumatic brain
injury or stroke and in chronic neurodegenerative diseases such as Alzheimer’s disease
and frontotemporal dementia. Therefore, individual values can only be interpreted within
the clinical context [7]. NfL measurements may also serve as a complementary tool to
expedite diagnosis and prognostication. For example, an elevated NfL level after a first
demyelinating event (RIS or CIS) could foreshadow a faster conversion to clinically definite
MS, potentially justifying earlier intervention [5]. Serum NfL (sNfL) has certain advantages
over traditional measures of MS disease progression such as MRI because it is relatively
noninvasive, inexpensive, and can be repeated frequently to monitor activity and treatment
efficacy. sNfL levels can be monitored regularly in patients with MS to determine change
from baseline and predict subclinical disease activity, relapse risk, and the development
of gadolinium-enhancing (Gd+) lesions [3]. However, inter-assay variability remains an
issue for sNfL. For example, a comparison of SiMoA vs. high-sensitivity ELISA for sNfL
found only a moderate correlation between platforms and significant biases in measured
levels [8]. This highlights the need to standardize NfL assay methods and reference ranges
across laboratories for consistent clinical implementation.

2.1.2. Tau Protein

Tau protein, which is commonly associated with Alzheimer’s disease, is also emerging
as a potential biomarker in MS. In MS, studies of CSF total tau (t-tau) have yielded mixed
results. Some patients with early or active MS exhibit mildly increased CSF tau compared
to controls, reflecting axonal damage, but levels are generally much lower than those
seen in primary neurodegenerative dementias [9]. The sensitivity of t-tau for MS is low to
moderate—many MS patients have normal CSF tau, especially outside of acute relapses [10].
Phosphorylated tau (p-tau), a hallmark biomarker in Alzheimer’s, does not typically show
a specific elevation in MS except in cases where an MS patient has coexisting Alzheimer
pathology. One study reported that a subset of progressive MS patients with severe
cognitive impairment had abnormal CSF p-tau and Aβ profiles, suggesting a concurrent
neurodegenerative process [10]. Thus, tau is more useful in ruling out other diagnoses than
as a primary MS marker [11,12]. Hyperphosphorylated tau species have also been detected
in MS and correlated with cognitive impairment and disease severity [13].

2.1.3. Amyloid—Precursor Protein (APP)

APP is produced by astrocyte cells during demyelination and can be located in reactive
glial cells during de- and remyelination. Higher APP levels are present in MS patients
and correlate with CNS lesion development [4]. In MS, APP is not measured in biofluids
as a soluble biomarker but examined in tissue as an indicator of acute axonal damage.
Histopathological studies have shown that transected axons in acute MS lesions often
exhibit accumulations of APP as a reaction to injury [14]. Gehrmann et al. first reported
APP expression within MS plaques, signifying ongoing axonal transection. The presence of
APP immunoreactive axonal spheroids in biopsy or autopsy specimens of MS confirms that
active demyelination is accompanied by neurodegeneration [15]. Thus, while APP servers
as a qualitative tissue biomarker of axonal damage in MS lesions, it is not used diagnosti-
cally in live patients. Its significance lies in the pathological evidence of neurodegeneration
in MS.
Diagnostics 2025, 15, 1178 5 of 18

2.1.4. Tubulin Beta (TUB β)

TUB β are heterodimeric proteins that make up microtubules. Neuron regeneration is
associated with increased production of the class ll tubulin isotype and increased in MS
patients compared to patients with other neurological diseases [4]. However, tubulin beta
is not a well-established MS biomarker at present. Some proteomic studies have identified
tubulin peptides in CSF when comparing MS patients to controls, suggesting that it is
part of a panel of injury-related proteins [14]. The sensitivity or specificity of tubulin beta
for MS has not been clearly reported in the literature, likely due to insufficient focused
studies. As a structural protein, tubulin is abundant in the CNS, but distinguishing whether
its presence in CSF is MS-specific or due to non-specific neural damage is challenging.
Therefore, tubulin beta remains an exploratory biomarker. It may be considered in future
panel assays or targeted analyses of neuronal exosomes, but, currently, it has no confirmed
clinical utility in MS.

2.2. Biomarkers of Neuronal Damage

2.2.1. 14-3-3 Protein
14-3-3 protein, which is present in neurons, can be measured in the CSF of both
patients with MS and those with Creutzfeldt–Jakob disease. However, the role of 14-3-3
protein in MS is inconsistent. Studies have found that 14-3-3 protein in the CSF is associated
with more severe disability, more extensive involvement of the spinal cord, and quicker
progression to MS or disease progression. The early accumulation of 14-3-3 protein in the
CSF may be related with decreased rates of recovery [4]. 14-3-3 is typically not detectable
in most MS patients, as the sensitivity of 14-3-3 overall is low (only a minority of MS cases
are positive), but, when present, it has a high specificity for significant CNS tissue damage
(since 14-3-3 rarely appears, except in conditions causing substantial neuronal death).
Therefore, a positive 14-3-3 in an MS patient could serve as a red flag for an atypically
aggressive course [4]. However, due to its low prevalence, 14-3-3 is not used as a routine
biomarker in MS.

2.2.2. Neuron Specific Enolase

NSE is a glycolytic enzyme found in neurons, and elevated levels in CSF or blood
indicate neuronal injury. In MS, NSE has been studied as a potential marker of neuroaxonal
damage. Research findings have shown that NSE levels are modestly higher in MS patients
compared to healthy controls, particularly during relapses or in progressive phases, though
the differences are not as pronounced as with NfL [14,16]. The sensitivity of NSE for
detecting MS activity is moderate; one large study found that serum NSE was not a
strong predictor of disability progression in relapsing MS, but, in primary progressive MS
patients, there was a moderate correlation between higher NSE levels and worse disability
scores [16]. Specificity is also moderate, since NSE can be elevated in various neurological
conditions (e.g., stroke, head injury). Overall, NSE alone appears to be a weaker biomarker
in MS than NfL. Its levels tend to increase with age and other factors, which confounds its
utility [16]. While elevated NSE in CSF may confirm the occurrence of neuronal damage
in MS (especially if other markers are unavailable), its clinical use is limited due to lower
sensitivity and specificity. Some authors have suggested NSE could be part of a panel of
biomarkers, but, on its own, it has not proven highly informative [12]. Concentrations of
neuron specific enolase (NSE), an enzyme found in neurons and axons that can be used to
estimate neuronal density, have been found to be increased in both the CSF and serum of
patients suffering from trauma, hypoxic brain injury, or cerebral bleeding. However, its
role in MS is rather inconsistent since several studies showed no difference between MS
patients and the control [4].
Diagnostics 2025, 15, 1178 6 of 18

2.3. Biomarkers of Glial Dysfunction

2.3.1. Glial Fibrillary Acidic Protein (GFAP)
GFAP is an astrocytic marker that reflects astrocyte activation and gliosis in MS. High
GFAP levels are associated with neuroinflammation and progressive MS phenotypes. This
biomarker is particularly useful in distinguishing MS subtypes. Recent ultrasensitive assays
have enabled the measurement of GFAP in serum; studies have found that serum GFAP is
higher in progressive MS patients compared to those with relapsing–remitting MS, and
higher GFAP associates it with more advanced disease [12,17]. GFAP appears to have
high specificity for progressive neurodegeneration in MS—in other words, elevated GFAP
is unlikely in purely inflammatory states without significant tissue injury [18]. Higher
serum and CSF GFAP levels were found to prognosticate worse outcomes of progression
as well as brain atrophy, especially in the gray matter and independent of serum NfL,
making it an important biomarker particularly because it is less associated with the acute
clinical phase of the disease. Serum GFAP levels tend to rise during relapses in relapsing–
remitting MS compared to periods of remission while being able to predict confirmed
disability progression even in non-active MS cases [7]. The current limitation is establishing
standardized reference ranges. Nonetheless, GFAP’s potential has led to its inclusion
in biomarker research panels and its consideration for use alongside NfL in managing
progressive MS.

2.3.2. Chitinase-3-like Protein 1 (CHI3L1, Also Known as YKL-40)

CHI3L1, also known as YKL-40, is a marker of glial activation and neuroinflammation.
Increased CSF CHI3L1 levels have been linked to a more severe disease course and worse
prognosis in MS patients. CHI3L1 has been proposed as a biomarker for distinguishing
patients with aggressive MS who may require early intervention with high-efficacy treat-
ments [12,14]. YKL-40 appears to reflect a component of the disease related to inflammation
and tissue remodeling by glia. In terms of performance, CSF YKL-40 has moderate sensitiv-
ity for MS disease activity (it may not rise during every relapse but tends to be higher in
patients with a more aggressive disease course) and moderate specificity (it is also elevated
in other neurological conditions involving neuroinflammation, such as CNS infections
or neurodegenerative diseases) [14]. However, some studies have identified YKL-40 as
an independent predictor of disability progression in MS, even when accounting for NfL
levels. This suggests that combining markers of axonal damage (NfL) with markers of glial
activation (YKL-40) could improve prognostic accuracy. Overall, YKL-40 is a promising
biomarker for the “slow burn” astroglial pathology in MS and may help identify patients
at the risk of progression. Ongoing research is working to validate cutoff values and to
standardize assays so that YKL-40 might be used clinically alongside NfL in the future.

2.3.3. S100B Protein

S100B is a calcium-binding protein produced predominantly by astrocytes (and to
some extent by oligodendrocytes) that serves as a marker of blood–brain barrier dysfunc-
tion and glial activation. Increased serum and CSF levels of S100B have been linked to
neuroinflammation and neurodegeneration in MS. Consequently, it has the potential to
serve as a useful biomarker for monitoring disease activity and treatment response [12].
CSF S100B levels have been found to be slightly elevated compared to controls, particularly
during active disease phases. The reported sensitivity of CSF S100B for MS is modest
(~65–75%), and specificity is also around 70–80% [7]. These values indicate that S100B alone
is not a strong discriminator, but elevated S100B does suggest the presence of CNS damage.
Some studies have linked higher S100B to gadolinium-enhancing lesions on MRI, implying
that it may reflect acute inflammation and BBB leakiness. However, because S100B can
Diagnostics 2025, 15, 1178 7 of 18

be elevated in many neurological conditions (including trauma and stroke), its specificity
for MS lesions is limited. In practice, S100B is not widely used as an MS biomarker, but
it continues to be studied. It might have a niche role in research settings to understand
glial responses or to be part of a broader biomarker index. For example, combining S100B
with other markers (like NSE or YKL-40) might increase overall sensitivity to detect any
neuroglial injury. At present, the clinical impact of S100B in MS is minor compared to more
robust markers like NfL or GFAP.

2.3.4. Chitotriosidase-1 (CHIT1)

Chitotriosidase-1 (CHIT1), a glycosyl hydrolase secreted predominantly by activated
microglia, has emerged as a robust biomarker candidate for predicting neurodegeneration
in multiple sclerosis (MS). Several high-quality studies have consistently demonstrated
the prognostic value of CSF CHIT1 levels. Oldoni et al. reported that elevated CSF CHIT1
at diagnosis significantly predicted greater long-term brain tissue damage and disability
accumulation in MS patients, distinguishing those with aggressive disease courses [19].
Supporting these findings, Beliën et al. analyzed a large cohort of 192 MS patients and
found that CSF CHIT1 was the strongest predictor of early EDSS progression among
myeloid biomarkers and histologically localized to lipid-laden microglia within active
demyelinating lesions [20]. Furthermore, Cross et al. demonstrated that higher baseline
CHIT1 levels correlated positively with the future development of slowly expanding lesions,
a marker of chronic active pathology [21]. Collectively, these studies establish CHIT1 as a
powerful biomarker reflecting early microglial activation and predicting disease severity,
with a strong potential for clinical translation into personalized MS monitoring strategies.

2.3.5. Soluble TREM2 (sTREM2)

Soluble TREM2 (sTREM2), the cleaved extracellular domain of the TREM2 receptor
expressed by microglia, has also been investigated as a marker of neuroinflammation
and neurodegeneration in MS. Initial studies conducted by Piccio et al. identified ele-
vated CSF sTREM2 levels in patients with active MS, correlating with markers of CNS
inflammation [22]. More recently, Ioannides et al. (2021) confirmed higher CSF sTREM2
concentrations in MS patients compared to healthy controls, with positive associations
observed between sTREM2, axonal injury markers (neurofilament light chain and phos-
phorylated neurofilament heavy chain), and disability scores such as EDSS and MSSS [23].
However, in contrast to CHIT1, sTREM2 did not consistently differentiate MS subtypes or
predict long-term progression. Genetic studies have also suggested a modest causal rela-
tionship between elevated sTREM2 levels and MS risk [24]. Overall, while sTREM2 reflects
ongoing microglial activation and appears mechanistically relevant to MS pathology, its
prognostic and diagnostic utility remains less definitive compared to CHIT1, necessitating
further large-scale longitudinal validation.

2.4. Biomarkers of Myelin Biology/Demyelination

Myelin Basic Protein
Myelin basic protein (MBP) is produced by the oligodendrocytes from the central
nervous system and has been found to be increased in the CSF of patients with MS. MS
patients with an acute exacerbation had higher levels than those with slower progressive MS
and even higher than those in remission [4]. MBP is a major constituent of the myelin sheath,
and, when myelin is destroyed, fragments of MBP can be released into the CSF. Historically,
CSF MBP was one of the earliest biochemical markers proposed to track demyelination
in MS. During acute MS relapses, especially those with substantial tissue damage, CSF
MBP levels can rise transiently. The sensitivity of CSF MBP for MS activity is limited—it
tends to be detectable only during acute phases in some patients and often returns to
Diagnostics 2025, 15, 1178 8 of 18

normal in remission. Its specificity for MS is also limited because any demyelinating
or CNS destructive process (e.g., traumatic brain injury, other demyelinating diseases)
can elevate MBP in CSF [25]. Clinical studies found that adding MBP measurement did
not significantly improve MS diagnostic accuracy when oligoclonal bands were already
considered [26,27]. As a result, MBP testing in CSF is now rarely used in MS management.
Nonetheless, MBP provides evidence of active myelin breakdown, and high MBP levels in
CSF correlate with severe attacks or acute disseminated encephalomyelitis. In summary,
while MBP is conceptually a direct marker of demyelination, its short-lived elevation and
poor disease specificity make it a less useful routine biomarker in MS (Table 1); (Figure 1).

Table 1. Sensitivity and specificity of current neurodegenerative biomarkers.

Evidence
Biomarker Specificity Sensitivity Cohort Reference
Strength
Bittner et al.
MS patients (serum,
(2021) [28]
n = 3028); multiple
Neurofilament Kapoor et al.
Moderate to High High studies including High
Light Chain (NfL) (2020) [29]
Bittner et al.
Fox et al.
(n = 683)
(2021) [30]
MS patients (CSF,
Ingram et al.
Total Tau (t-Tau) Low to Moderate Variable n = 990) vs. controls Moderate
(2010) [9]
(n = 615)
RRMS and PMS
Glial Fibrillary
patients (CSF, Chitnis et al.
Acidic Protein High Moderate High
n = 165); Chitnis (2024) [17]
(GFAP)
et al. cohort
MS patients (CSF,
Moderate Moderate Di Filippo et al.
S100B n = 120); Moderate
(Exploratory) (Exploratory) (2024) [7]
single-center cohort
RRMS, SPMS, PPMS
Comabella
Neuron-Specific patients (CSF,
Low to Moderate Low to Moderate et al. (2010) Moderate
Enolase (NSE) n = 250); pooled
[14]
cohort analysis
High Sensitivity CIS patients (n = 21); Colucci et al.
in CIS; RRMS/SPMS (2004); [31]
14-3-3 Protein High in CIS Moderate
Limited Specificity (n = 39); Colucci et al. Sèze et al.
in MS single-center study (2002) [32]

Neurodegenerative Biomarkers
Future Biomarkers:

2.5. Metabolomic Biomarker

The field of metabolomics, which involves the study of metabolites in biofluids dur-
ing disease states, is emerging as a powerful approach, as distinct metabolite signatures
could be a potential biomarker for disease progression or predictive of the beneficial
effect of DMTs in MS. Few studies have outlined a distinct metabolic signature, includ-
ing serum phospholipids, altered bile acid metabolism, abnormalities in aromatic amino
acid metabolism, and pro-resolving lipid mediators in MS compared to healthy subjects,
which could be developed as a biomarker for disease and/or novel therapy [4,33]. Some
metabolite profiles appear to distinguish progressive MS from relapsing MS or from
healthy controls. The sensitivity of individual metabolites is typically low, but, as a
Diagnostics 2025, 15, 1178 9 of 18

panel, metabolomic signatures can achieve useful accuracy. One emerging metabolite
of interest is N-acetylaspartate (NAA)—traditionally measured by MR spectroscopy as a
marker of neuronal integrity; reduced NAA in CSF could reflect neuronal loss (perhaps
indirectly measurable via metabolomics assays). Another example is a pattern of altered
tryptophan metabolism, including the kynurenine pathway, which metabolomic analysis
can capture [34]. While metabolomics offers a broad view, translating this into a practi-
cal biomarker test is challenging. It may be that, in the future, a combination of a few
key metabolites will be formulated into a multiplex biomarker panel for MS. As of now,
Diagnostics 2025, 15, x FOR PEER REVIEW 9 of 18
metabolomic biomarkers for MS are in the discovery phase, lacking validation for routine
clinical use.

1. Specificity
Figure 1.
Figure Specificityand
andsensitivity of of
sensitivity neurodegenerative biomarkers
neurodegenerative in multiple
biomarkers sclerosis,
in multiple including
sclerosis, in-
cohort sizes.
cluding cohort sizes.
2.5.1. Kynurenines
 Future Biomarkers (in early research phase; not yet clinically validated):
The kynurenine pathway is involved in neuroinflammation and excitotoxicity.
Kynurenic acid,Biomarker
2.5. Metabolomic a neuroprotective metabolite, is reduced in MS, whereas quinolinic acid,
a neurotoxic metabolite, is increased. This imbalance contributes to axonal damage and
The field of metabolomics, which involves the study of metabolites in biofluids
mitochondrial dysfunction, making kynurenines promising biomarkers for disease progres-
during disease states, is emerging as a powerful approach, as distinct metabolite signa-
sion and therapeutic targeting [35]. Alterations in this pathway could reflect the balance of
tures could be a potential biomarker for disease progression or predictive of the benefi-
neurodegeneration and neuroprotection. Studies indicate that MS patients often have an
cial effect of DMTs in MS. Few studies have outlined a distinct metabolic signature, in-
imbalance in kynurenine metabolites; for instance, increased levels of QUIN (a marker of
cluding serum phospholipids, altered bile acid metabolism, abnormalities in aromatic
macrophage/microglia activation) have been observed in MS lesions and CSF [34]. On the
amino acid metabolism, and pro-resolving lipid mediators in MS compared to healthy
other hand, levels of KYNA may be reduced in progressive MS, potentially diminishing
subjects, which could be developed as a biomarker for disease and/or novel therapy [4,
neuroprotection [36]. The sensitivity and specificity of kynurenine pathway metabolites
33]. Some metabolite profiles appear to distinguish progressive MS from relapsing MS or
as biomarkers are still being evaluated. They are not disease-specific (these metabolites
from healthy controls. The sensitivity of individual metabolites is typically low, but, as a
can be altered in other inflammatory neurological disorders), but a distinctive pattern such
panel, metabolomic signatures can achieve useful accuracy. One emerging metabolite of
as a high QUIN:KYNA ratio might be indicative of active MS pathology [34]. Overall,
interest is N-acetylaspartate (NAA)—traditionally measured by MR spectroscopy as a
kynurenines are promising research biomarkers that link neuroinflammation to neurode-
marker of neuronal integrity; reduced NAA in CSF could reflect neuronal loss (perhaps
generation; they could eventually complement protein biomarkers by providing metabolic
indirectly measurable via metabolomics assays). Another example is a pattern of altered
insight. However, current data on their diagnostic performance in MS are limited, and
tryptophan metabolism, including the kynurenine pathway, which metabolomic analysis
assays are not yet standardized for clinical use.
can capture [34]. While metabolomics offers a broad view, translating this into a practical
biomarker test is challenging. It may be that, in the future, a combination of a few key
metabolites will be formulated into a multiplex biomarker panel for MS. As of now,
metabolomic biomarkers for MS are in the discovery phase, lacking validation for routine
clinical use.

2.5.1. Kynurenines
Diagnostics 2025, 15, 1178 10 of 18

2.5.2. Lipid Biomarkers (Ceramides, Cholesterol, Oxysterols, Phospholipids)

Lipid metabolism plays a crucial role in myelin integrity and neuronal function.
Specific sphingolipids and phospholipids have been identified as potential biomarkers
for MS progression. Lipidomic profiling can help detect early neurodegenerative changes
and assess the impact of therapeutic interventions on lipid metabolism [37]. Lipids are
fundamental to myelin and cell membranes, and lipid disturbances have been observed in
MS. Demyelination releases cholesterol and other lipids, which are then metabolized into
compounds that can be detected in blood or CSF. Certain ceramides and sphingolipids have
been found at different levels in MS patients versus controls, implicating these molecules
as potential biomarkers of neurodegeneration [38]. For instance, elevated levels of specific
ceramide species were associated with more severe disability in some MS cohorts [39].
Oxysterols (oxidized cholesterol derivatives) like 24S-hydroxycholesterol are higher when
active demyelination occurs, reflecting CNS cholesterol turnover. Phospholipid profiles
in CSF have also been correlated with progressive MS. The sensitivity of individual lipid
markers varies, but, collectively, a lipid panel might capture pathological processes such
as demyelination or remyelination attempts. Neurodegenerative diseases, MS exhibits
a unique lipid signature, though not as pronounced as in primary dementias. Lipid
biomarkers also tie into other pathways; for example, high levels of certain lysosomal lipids
could indicate microglial activation. The specificity of lipid changes to MS is moderate—
many lipid alterations are seen in other neurodegenerative disorders—but combining them
with MS-specific clinical context enhances relevance. The use of lipid biomarkers is not
yet mainstream in MS, but they represent an emerging area. In the future, tracking lipid
changes could complement protein biomarkers, giving a fuller picture of tissue breakdown
and repair. For now, they remain largely research tools, requiring more evidence to become
part of clinical monitoring.

2.5.3. Extracellular Vesicles (EVs)

EVs can be broken down into microvesicles and exosomes based on size. Microvesicles
typically range from 100 to 1000 nanometers (nm), while exosome sizes generally fall within
the range of 50 to 150 nm. Exosomes have the capacity to facilitate communication between
cells and travel large distances in the body. Thus, they can be used as biomarkers for
monitoring MS disease progression and activity as well as therapeutic treatment. Exosomes
can be released from T cells to regulate antigen-presenting cells via miRNAs contained
within the exosome and act as proinflammatory regulators in rheumatoid arthritis, Grave’s
disease, and in MS. EVs are found to be increased in the CSF and plasma of MS patients
and have different molecular compositions compared to EVs from healthy individuals [4].
One study found that levels of EVs expressing astrocytic or neuronal markers were higher
during MS relapses and correlated with new MRI lesions, suggesting that EV counts could
reflect active disease [40]. The sensitivity of EV-based biomarkers can be high when using
advanced detection methods, because even subtle ongoing damage might continuously
release vesicles. Specificity depends on identifying cell-specific cargo, for example, EVs
carrying GFAP or neurofilaments would be more specific to CNS damage. Early results
are promising; elevated EVs originating from endothelial cells have been noted in MS
and linked to blood–brain barrier breakdown, whereas EVs containing synaptic proteins
might indicate neurodegeneration [41]. However, EV analysis is technically complex and
not yet standardized. The clinical utility remains exploratory—EV measurements could
one day become a liquid biopsy of the CNS, offering a composite view of various cell
injuries in MS. Currently, they provide research insights and may improve our under-
standing of MS pathology and intercellular communication, rather than serving as routine
clinical biomarkers.
Diagnostics 2025, 15, 1178 11 of 18

The following table reunites both actual and future possible biomarkers and their
current and possible clinical importance development in MS patients (Table 2).

Table 2. Current and future possible neurodegenerative biomarkers regarding clinical utility.

Biomarker Biological Sample Significance in MS Clinical Utility Clinical Status References

CURRENT BIOMARKERS
Biomarker of axonal Khalil et al.
Neurofilament Diagnostic and
damage; correlates 2024; [6]
Light CSF, Blood Progression Approved
with disease activity Bittner et al.
Chain (NfL) Monitoring
and progression. 2021 [28]
Implicated in
Tau Protein neurodegeneration Progression Under Londoño et al.
CSF
(t-tau, p-tau) and cognitive Monitoring Validation 2022 [10]
dysfunction in MS.
Associated with
severe disability and
Progression Under Colucci et al.
14-3-3 Protein CSF faster MS
Monitoring Validation 2004 [31]
progression;
limited sensitivity.
Elevated levels
Neuron-Specific during progression Progression Under Koch et al.
CSF, Blood
Enolase (NSE) phases; neuronal Monitoring Validation 2015 [16]
damage marker.
Sandi et al.
Kynurenines Imbalance affects
Progression 2021; [34]
(Kynurenic Acid, CSF, Blood neuroinflammation Exploratory
Monitoring Pukoli et al.
Quinolinic Acid) and neurotoxicity.
2021 [36]
Amyloid Marker of acute Diagnostic and Gehrmann
Under
Precursor CSF, Brain Tissue axonal injury during Progression et al. 1995
Validation
Protein (APP) demyelination. Monitoring [15]
Associated with
Tubulin Beta neuron regeneration; Progression Comabella
CSF, Brain Tissue Exploratory
(TUB β) elevation seen Monitoring et al. 2010 [14]
in MS.
Reflects acute
Diagnostic and Ziemssen
Myelin Basic demyelination; Under
CSF Progression et al.
Protein (MBP) increased Validation
Monitoring 2019 [25]
during relapses.
Associated with Momtazmanesh
Chitinase-3-like
glial activation Progression Under et al. 2021 [12];
Protein 1 CSF
and aggressive Monitoring Validation Comabella
(CHI3L1/YKL-40)
MS forms. et al., 2010 [14]
Chitnis et al.
Marker of
Glial Fibrillary 2024 [17];
astrocytic activation; Progression Under
Acidic CSF, Blood Momtaz-
correlates with Monitoring Validation
Protein (GFAP) manesh et al.
progressive MS.
2021 [12]
Marker of
blood–brain barrier Progression Under Di Filippo
S100B Protein CSF, Blood
dysfunction; modest Monitoring Validation et al., 2024 [7]
diagnostic utility.
Diagnostics 2025, 15, 1178 12 of 18

Table 2. Cont.

Biomarker Biological Sample Significance in MS Clinical Utility Clinical Status References

Marker of microglial
Oldoni et al.
activation; strong
Chitotriosidase-1 Prognostic Under 2020 [19];
CSF predictor of disease
(CHIT1) Monitoring Validation Beliën et al.
severity and
2024 [20]
brain atrophy.
Reflects microglial
activation and Ioannides
Soluble TREM2 axonal injury; Progression et al. 2021 [23];
CSF Exploratory
(sTREM2) prognostic Monitoring Piccio et al.
associations 2008 [22]
suggested.
FUTURE BIOMARKERS
Manna et al.
Altered in
Diagnostic and 2024 [40];
Extracellular MS; potential
CSF, Blood Progression Exploratory Gutiérrez-
Vesicles (EVs) for monitoring
Monitoring Fernández
disease activity.
et al. 2021 [41]
Metabolomics Emerging metabolic
Diagnostic and
(General markers linked to Singh et al.
Blood, CSF Progression Exploratory
Metabolite progression and 2019 [33]
Monitoring
Panels) therapy response.
Disruption in Mey et al.
Kynurenines
kynurenine pathway Progression 2023 [35];
(Detailed CSF, Blood Exploratory
contributes to Monitoring Sandi et al.
Pathway)
neurodegeneration. 2021 [34]
Lipid
Changes in lipid
Biomarkers
composition Diagnostic and Wei et al.
(Ceramides,
Blood, CSF associated with Progression Exploratory 2024 [38]; Jiao
Cholesterol,
myelin integrity and Monitoring et al. 2024 [37]
Oxysterols,
neurodegeneration.
Phospholipids)

CSF vs. Blood-Based Biomarkers: Comparison and Clinical Applications

Cerebrospinal fluid (CSF) has traditionally been the fluid of choice for CNS biomark-
ers because of its proximity to the brain and spinal cord. CSF levels of neurodegenerative
markers are often higher and more directly reflect CNS pathology, making them very
sensitive in research settings [42]. For example, before ultrasensitive assays existed, NfL
and other proteins were reliably measurable only in CSF. Consequently, an analysis of CSF
can yield highly precise insights into neuronal damage [42]. However, lumbar puncture to
obtain CSF is invasive and not practical for frequent monitoring. The discomfort experi-
enced by patients, in addition to the inherent risks associated with the procedure, mean
CSF biomarkers are typically checked only at diagnosis or in research, rather than serially
in routine care [42].
Blood-based biomarkers overcome this limitation. These biomarkers have the advan-
tages of being low risk and inexpensive, allowing repeat measurements to track disease
course [42]. The main advance enabling blood-based neurodegeneration markers was the
development of highly sensitive immunoassays, which has opened new opportunities
in MS biomarker utilization—making markers once limited to CSF quantified in blood
and accessible for clinical monitoring (e.g., SIMOA) [5]. For instance, serum NfL closely
Diagnostics 2025, 15, 1178 13 of 18

mirrors CSF NfL and has become the first blood biomarker to show solid utility in MS [43].
Blood tests for NfL are being pilot-tested in clinics to inform treatment decisions, and
professional guidelines are beginning to consider how to incorporate sNfL into practice
(e.g., proposing cutoff values by age) as a tool to monitor MS evolution. The advantages
of blood biomarkers are clear: they enable the high-frequency tracking of disease activ-
ity/progression, potentially even at every clinical visit, and can be more cost-effective for
long-term follow-up [42]. This is particularly useful for assessing treatment response or
catching early signs of progression without requiring an MRI each time [30]. Addition-
ally, blood biomarkers can be included in large trials or longitudinal studies with ease,
facilitating research on neuroprotective strategies.
Limitations: Despite their convenience, blood-based markers have some disadvan-
tages. Concentrations in blood are much lower than in CSF, so results can be influenced by
assay precision and variability. There is also the issue of biological noise—blood values
might be affected by peripheral factors and are not entirely specific to MS; any significant
neuronal damage (trauma, stroke, other neurodegeneration) can raise NfL, and it must be
interpreted within context [5]. CSF, by contrast, is a more specific matrix for CNS changes
(fewer confounding peripheral sources), but, again, routine CSF draws are impractical.
Thus, each has its role: CSF biomarkers excel in diagnostic contexts or one-time assessments
(such as confirming MS and gathering a baseline profile of CNS injury, including both
neurodegenerative and inflammatory indices), whereas serum biomarkers are ideal for
ongoing monitoring and have clear potential in the day-to-day clinical management of MS.
Currently, the most common CSF test for MS diagnosis is the IgG oligoclonal band assay,
highlighting that neurodegeneration markers in CSF are not yet standard of care [30]. It is
worth noting that no biomarker alone dictates management; rather, these tests add a layer
of evidence. For instance, elevated serum NfL levels may prompt the consideration of more
rapid MRI scans or a re-evaluation of therapeutic interventions. As research progresses
and standard reference ranges are defined, we can expect serum-based neurodegenera-
tive biomarkers (like NfL, GFAP) to become part of routine MS care for tracking disease
progression and treatment effectiveness, while CSF-based measures are more efficient for
certain diagnostic or research applications where maximum specificity is required [5].

3. Expanding Clinical Applications of Biomarkers in MS

Biomarkers play a pivotal role in clinical decision making, allowing for enhanced
disease monitoring, treatment response assessment, and personalized medicine approaches.
Their integration into routine practice could revolutionize MS management.

3.1. Diagnosis and Early Detection

Neurodegenerative biomarkers, particularly neurofilament light chain (NfL), have
demonstrated high sensitivity in detecting axonal injury at early disease stages [6,13]. Ele-
vated serum NfL levels can differentiate MS from other neurological disorders, providing
a non-invasive diagnostic tool [28]. When combined with oligoclonal bands (OCBs) in
cerebrospinal fluid, these markers improve diagnostic accuracy [11].
Furthermore, advanced multi-omics approaches integrating transcriptomics, pro-
teomics, and metabolomics are emerging as powerful diagnostic tools. Studies suggest
that combining NfL with genetic and metabolic markers could improve early detection
accuracy, particularly in individuals at risk of developing MS.

3.2. Disease Progression Monitoring

Regular monitoring of NfL levels in serum or cerebrospinal fluid provides real-time
insights into ongoing neurodegeneration [44]. Increased levels correlate with brain atrophy
Diagnostics 2025, 15, 1178 14 of 18

and worsening disability, making it a valuable biomarker. Additionally, lipidomic profiling

can help identify metabolic changes associated with worsening symptoms [37].
Continuous biomarker assessment, including neuroinflammatory cytokines and glial
activation markers, could aid disease tracking. Longitudinal studies demonstrate that dy-
namic changes in these biomarkers can predict relapses and progressive disease transition,
enabling early therapeutic interventions.

3.3. Prognostication and Risk Stratification

Kynurenine pathway metabolites, including quinolinic acid, serve as prognostic
biomarkers by identifying patients at higher risk for aggressive disease progression [35]. El-
evated levels of GFAP and CHI3L1 have also been linked to poorer long-term outcomes [12].
These biomarkers help in risk stratification and guide treatment intensity decisions, ensur-
ing that patients receive the most appropriate and effective treatment for their condition.
Emerging research has explored the potential of gut microbiota-derived metabolites
as indicators of MS progression. Alterations in gut microbial composition and associated
metabolite levels have shown promise in distinguishing between relapsing and progressive
disease phenotypes.

3.4. Treatment Response Evaluation

Serum NfL has emerged as a reliable biomarker for assessing treatment efficacy [28].
Patients on high-efficacy disease-modifying therapies (DMTs) such as natalizumab and
ocrelizumab exhibit significantly lower NfL levels over time, indicating reduced neuroax-
onal damage [13]. Monitoring lipid biomarkers can also provide insights into the metabolic
impact of DMTs [37].
Artificial intelligence (AI)-driven biomarker analysis is an evolving field that enables
personalized therapeutic adjustments. AI models trained on biomarker datasets can predict
treatment responsiveness, reducing trial-and-error in therapy selection.

3.5. Personalized Treatment Strategies

The integration of multiple biomarkers allows for a precision medicine approach
in MS management [11]. By combining CSF, blood-based, and molecular biomarkers
with machine learning algorithms, clinicians can predict individual responses to specific
therapies, minimizing adverse effects and optimizing outcomes [37].
Precision medicine approaches now extend to biomarker-guided drug repurposing
exploring whether existing medications for neurodegenerative diseases can be repurposed
for MS based on shared biomarker profiles (Figure 2).

3.6. Expanding Biomarker Research in MS

The future of MS biomarker research focuses on developing multi-biomarker panels
that integrate the following:
• Neuroaxonal Injury Markers: NfL, tau protein, and neurogranin.
• Glial Activation Markers: GFAP, CHI3L1, and S100B.
• Neuroinflammatory Markers: Cytokine panels, complement proteins.
Metabolomic Markers: Kynurenines, lipidomic profiles, gut microbiome metabolites.
 machine learning algorithms, clinicians can predict individual responses to specific
therapies, minimizing adverse effects and optimizing outcomes [37].
Precision medicine approaches now extend to biomarker-guided drug repurposing
Diagnostics 2025, 15, 1178 exploring whether existing medications for neurodegenerative diseases can be 15
repur-
of 18
posed for MS based on shared biomarker profiles (Figure 2).

Figure2.
Figure 2. Emerging
Emerging biomarkers
biomarkersand
andclinical
clinicalpractice.
practice.

4.
3.6.Discussion
Expanding Biomarker Research in MS
This reviewofhighlights
The future the growing
MS biomarker researchutility
focusesof on
neurodegenerative biomarkers panels
developing multi-biomarker in im-
proving diagnostic
that integrate precision, tracking disease progression, and optimizing therapeutic
the following:
strategies in multiple sclerosis (MS). The evidence strongly supports the clinical value of
serum and CSF neurofilament light chain (NfL), particularly for monitoring axonal damage
and treatment response. Emerging biomarkers, such as glial fibrillary acidic protein (GFAP),
chitinase-3-like protein 1 (CHI3L1), and metabolomic indicators, demonstrate promise in re-
fining our understanding of progressive MS phenotypes and tailoring patient-specific care.
However, several challenges remain. Many emerging biomarkers still lack standard-
ized reference ranges, robust longitudinal data, and cross-cohort validation. Interindividual
variability, non-specificity to MS, and assay-dependent differences may also confound
results. Despite these challenges, integrating multi-biomarker panels—encompassing neu-
roaxonal, glial, and metabolic indicators—offers a future framework for precision neurology
in MS care. The use of biomarkers should complement, not replace, clinical judgment and
imaging tools. Further research is necessary to validate these biomarkers across diverse
populations and establish consensus guidelines for their routine use.

5. Limitations of This Review

This review is narrative in nature and does not include a formal meta-analysis, which
may limit the statistical rigor of conclusions. Although comprehensive, the study selection
process was not blinded, and publication bias may have influenced the inclusion of more
well-known or favorable biomarker studies. Additionally, the heterogeneous nature of
included studies—with differences in patient populations, assay techniques, and biomarker
thresholds—limits direct comparisons. Finally, rapid advancements in biomarker technolo-
gies and bioinformatics mean that the field is evolving, and newer markers may soon alter
current clinical paradigms.
Diagnostics 2025, 15, 1178 16 of 18

6. Conclusions
Neurodegeneration significantly contributes to MS-related disability, underscoring
the need for reliable biomarkers to monitor disease progression. CSF and blood-based
biomarkers, in conjunction with advanced imaging modalities, offer promising paths for
early diagnosis, prognostic evaluation, and treatment response evaluation. The analysis of
biomarkers such as NfL, GFAP, CHI3L1, tau protein, kynurenines, and lipid markers pro-
vide valuable insights into disease evolution. Future research should focus on integrating
these biomarkers into clinical practice, developing multi-biomarker panels. By advancing
neurodegenerative biomarker research, clinicians will be better equipped to personalize
treatment strategies and improve long-term disease management in MS.

Author Contributions: Conceptualization, A.C., M.I.S. and E.B.I.; methodology, D.A.; software, C.G.;
validation, R.C., M.I.S. and R.-C.A.; formal analysis, A.C.; investigation, I.P.; resources, A.I.C.; data
curation, A.M.B.; writing—original draft preparation, M.I.S.; writing—review and editing, A.C.;
visualization, C.M.G.; supervision, E.B.I.; project administration, C.G.; funding acquisition, D.A. All
authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The original contributions presented in this study are included in the
article. Further inquiries can be directed to the corresponding author(s).

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

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