Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 2, pp. 377-391 © Pleiades Publishing, Ltd., 2024.

377

REVIEW

Developing Peripheral Biochemical Biomarkers

of Brain Disorders: Insights from Zebrafish Models

Nikita P. Ilyin

1,a

, Elena V. Petersen

2,b

, Tatyana O. Kolesnikova

3,c

Konstantin A. Demin

1,2,4,5,d

*, Sergey L. Khatsko

6,e

, Kirill V. Apuhtin

7,8,f

and Allan V. Kalueff

1,4,6,7,g

Institute of Translational Biomedicine, St. Petersburg State University, 199034 St.Petersburg, Russia

Moscow Institute of Physics and Technology, 115184 Moscow, Russia

Neuroscience Program, Sirius University of Science and Technology, 354340 Sochi, Russia

Institute of Experimental Medicine, Almazov National Medical Research Centre,

Ministry of Healthcare of the Russian Federation, 197341 St.Petersburg, Russia

Laboratory of Preclinical Bioscreening, Granov Russian Research Center of Radiology and Surgical Technologies,

Ministry of Healthcare of the Russian Federation, 197758 Pesochny, Russia

Ural Federal University, 620002 Ekaterinburg, Russia

Laboratory of Biopsychiatry, Scientific Research Institute of Neurosciences and Medicine,

630117 Novosibirsk, Russia

Neuroscience Division, Sirius University of Science and Technology, 354340 Sirius Federal Territory, Russia

e-mail: nik.ilyn.98@gmail.com

e-mail: petersen.ev@mipt.ru

e-mail: kolesnikova.to@talantiuspeh.ru

e-mail: k.demin@spbu.ru

e-mail: sergey.khatsko@urfu.ru

e-mail: kirillapuhtin@mail.ru

e-mail: avkalueff@gmail.com

Received August 22, 2023

Revised January 9, 2024

Accepted February 13, 2024

Abstract— High prevalence of human brain disorders necessitates development of the reliable peripheral bio-

markers as diagnostic and disease-monitoring tools. In addition to clinical studies, animal models markedly ad-

vance studying of non-brain abnormalities associated with brain pathogenesis. The zebrafish (Danio rerio) is be-

coming increasingly popular as an animal model organism in translational neuroscience. These fish share some

practical advantages over mammalian models together with high genetic homology and evolutionarily conserved

biochemical and neurobehavioral phenotypes, thus enabling large-scale modeling of human brain diseases. Here,

we review mounting evidence on peripheral biomarkers of brain disorders in zebrafish models, focusing on al-

tered biochemistry (lipids, carbohydrates, proteins, and other non-signal molecules, as well as metabolic reactions

and activity of enzymes). Collectively, these data strongly support the utility of zebrafish (from a systems biology

standpoint) to study peripheral manifestations of brain disorders, as well as highlight potential applications ofbio-

chemical biomarkers in zebrafish models to biomarker-based drug discovery and development.

DOI: 10.1134/S0006297924020160

Keywords: zebrafish, brain disorders, biomarker, peripheral, neurodegeneration, stress, epilepsy

* To whom correspondence should be addressed.

INTRODUCTION

Central nervous system (CNS) disorders, such as

neurodegenerative (Alzheimer’s and Parkinson’s) and

affective (depression and anxiety) diseases, are com-

plex, wide-spread, and treatment-resistant, and they

contribute significantly to the global public health

costs [1, 2]. The lack of objective evidence-based ap-

proaches, especially for early diagnostics of these

disorders, underlies the growing interest to studying

biomarkers of the CNS pathogenesis, including be-

havioral, morphological, and molecular responses.

ILYIN et al.378

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Abbreviations: ATP,adenosine triphosphate; CNS,central nervous system; EEG,electroencephalography; GABA,γ-amino-

butyric acid; NPC,Niemann–Pick typeC; PLP,pyridoxal 5′-phosphate; PLPBP, PLP-binding protein; PNPO,pyridoxamine

5′-phosphate oxidase.

In addition to molecular biomarkers in the brain,

pathological biochemical changes also occur peripher-

ally, necessitating identification of novel biochemical

biomarkers of CNS disorders [3, 4].

Studying clinical biomarkers in vivo is often hin-

dered by impossibility of measuring pathological

markers directly in the brain tissue using invasive

methods. Thus, using pathological biomarkers from

peripheral samples, such as blood or skin, becomes

more relevant [3, 5]. Validity of this approach is based

on the fact that various signs of central pathology can

be successfully measured in peripheral tissues. For ex-

ample, while this is particularly common for the dis-

eases associated with neuroendocrine deficits or with

damaged blood-brain barrier (BBB) [6], peripheral bio-

markers can originate in the periphery, reflecting sys-

temic nature of CNS pathogenesis [7, 8].

In addition to the established clinical biomarkers,

there are also animal models, especially rodents, wide-

ly used to study CNS pathogenesis and biomarker val-

idation [9-11]. Because collecting brain tissue is not a

problem in animals, their peripheral biomarkers as-

sociated with central neuropathology are relatively

understudied. However, validity of the peripheral bio-

markers in animals would be markedly reinforced by

showing that they reproduce the signs of peripheral

dysregulations observed in clinical studies, e.g., endo-

crine hyperactivation in animal models of depression

[11, 12]. Moreover, animal models of CNS diseases can

help not only to identify peripheral biomarkers among

those already discovered in patients, but also to search

for novel biomarkers not yet identified previously [13].

Thus, studying non-brain abnormalities in animal mod-

els of CNS pathologies helps elucidate their pathophys-

iological mechanisms, providing novel insights into a

complex ‘systems biology’ nature of these disorders,

eventually facilitating translational research and de-

velopment of novel therapies [14, 15].

Addressing this problem further, here we re-

view mounting evidence on peripheral biomarkers

of brain disorders, focusing on altered biochemistry

(lipids, carbohydrates, proteins, and other non-sig-

nal molecules, as well as metabolic reactions and ac-

tivity of enzymes), in a novel model organism, the

zebrafish (Danio rerio). Zebrafish is becoming one of

the most important model species in biomedicine, in-

cluding neuroscience[16-20]. The idea for the present

paper originated from the invited talk on this topic,

given bythe senior co-author in May 2022, and subse-

quent discussion, at Professor Vladimir P. Skulachev’s

seminar at Belozersky Institute of Physico-Chemi-

cal Biology in Lomonosov Moscow State University.

Thispaper is dedicated to the memory of academician

V. P. Skulachev (1935-2023), a brilliant scientist, a re-

spected scholar, and a great colleague.

ZEBRAFISH MODELS

AND BIOMARKERS OF CNS DISORDERS

A common problem of using animal biomarkers

in biomedical research is their ambiguous interpreta-

tion and often somewhat unclear validity [21]. For ex-

ample, this problem is particularly inherent in transla-

tional neuroscience since various animal models may

have their own, acquired through natural evolution,

unique characteristics [22]. Thus, one of the strategies

for a more accurate and comprehensive modeling of

CNS pathobiology is to expand the number of model

objects (beyond the traditionally used rodents), there-

by utilizing a wider spectrum of animals from various

taxa [22-24]. A small teleost fish, the zebrafish, has re-

cently emergedas a novel powerful model organism in

translational neuroscience [25, 26]. This fish possesses

several key advantages over other model organisms

(e.g., rodents), providing a reasonable compromise

between the system complexity and practical sim-

plicity [27], and also enabling a large-scale modeling

of “core”, evolutionary conserved aspects of complex

brain disorders [28, 29]. Table 1 summarizes both ad-

vantages and limitations of zebrafish models in bio-

medical research (also see further).

Multiple zebrafish models of brain disorders have

also been developed (Tables 2 and 3), based on targeting

a wide range of their CNS biomarkers. For example,

similarly to rodent studies, zebrafish models of Parkin-

son’s disease utilize reduced brain levels of dopamine

and tyrosine hydroxylase (the main enzyme of dopa-

mine biosynthesis) as markers of dopaminergic neu-

ronal loss [43, 44]. Likewise, in the zebrafish models

of pentylenetetrazol (PTZ)-induced epilepsy, the brain

gene expression level of c-fos is often used as a bio-

marker of overall neuronal activity [45, 46]. Another

example involves biomarkers of neuroinflammation

observed in various zebrafish models of traumatic

brain injury [47, 48]. While multiple other (e.g., endo-

crine, genomic, and proteomic) peripheral biomarkers

of CNS disorders have already been developed as well,

here we focus on purely biochemical biomarkers, in-

cluding lipids, non-signaling proteins, carbohydrates,

amino acids and their derivatives that can be easily

analyzed in peripheral samples. Accordingly, descrip-

tion of the peripheral genomic (mutations and genet-

ic polymorphism), transcriptomic (genes expression

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BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Table 1. Brief summary of selected major advantages and limitations of utilizing zebrafish models in translational

biomedicine and neuroscience

Advantages References

Low cost of maintenance [30]

Rapid development [25]

Highly tractable genome [31]

High percent of genetic homology to humans [32]

Ease of pharmacological manipulations [30]

Ease of genetic manipulations [33]

Potential for high-throughput drug screening [34]

Limitations

Evolutionary remoteness from humans, contributing to some existing genetic, biochemical,

and physiological differences

[35, 36]

Certain neuroanatomical differences from mammals (e.g., lack of neocortex and midbrain

dopaminergic neurons)

[16, 37, 38]

Some of the genes exist in two copies compared to one copy in mammals

(due to the teleost-specific whole-genome duplication event)

[39]

Limited availability of the genetically characterized inbred and outbred strains compared

to rodent models

[26, 40]

Superior neuroregenertative potential (vs. mammals) that complicates modeling

neurodegenerative disorders

[41]

Lack of certain complex behaviors characteristic of mammals (e.g., parental care) [42]

Table 2. Selected examples of zebrafish models of human

brain disorders and their CNS biomarkers

Models References

Parkinson’s disease

↓ Tyrosine hydroxylase [43]

↓ Dopamine [44]

Epilepsy

↓ c-fos expression [45, 46]

↓ γ-Aminobutyric acid (GABA) [49]

Stress-related (affective) disorders

↓ Glucocorticoid receptor expression [50]

↓ Serotonin [51]

levels), and endocrine (hormones, cytokines) biomark-

ers was beyond the scope of the present study.

PERIPHERAL BIOCHEMICAL BIOMARKERS

OF ZEBRAFISH CNS PATHOGENESIS

Stress-related disorders. Stress is a key risk fac-

tor in pathogenesis of multiple CNS diseases, including

anxiety and depression – the two most common and

widespread brain maladies [76-78]. There are many ze-

brafish models based both on acute [79-82] and chron-

ic stress exposure [50, 83-89] that recapitulate the signs

of central and peripheral dysregulation characteristic

of stress-related pathologies in humans. While cortisol

is widely accepted as a peripheral biomarker of stress

level in zebrafish [90-92], metabolomic analyses may

also accurately assess the overall physiological stress

response of an organism [52].

Indeed, fish subjected to acute netting stress and

two different behavioral tests demonstrated changes

ILYIN et al.380

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Table 3. Zebrafish models of human brain disorders and their peripheral biochemical biomarkers

Models, biomarkers Responses References

Stress-related disorders

Alanine, taurine, adenosine, creatine, lactate, acetate, leucine/isoleucine,

and histidine

[52]

HADHB, hspa8, hspa5, actb1, mych4, atp2a1, zgc:86709, and zgc:86725 proteins ↑ [53]

Heart proteins of glucose metabolism, gluconeogenesis,

the ubiquitin–proteasome system and peroxisomal proliferator-activated

receptor signaling

↓ rate of synthesis [54]

Niemann–Pick type C disease (NPC)

Unesterified cholesterol ↑ [55-59]

Phospholipids and sphingolipids

altered

whole-body levels

[60]

Epilepsy (metabolic biomarkers)

Glycolysis rate and mitochondrial respiration rate ↓ [61-63]

Basal respiration, maximal respiration, mitochondrial respiration,

proton leaks, and ATP-linked respiration rates

↑ [64]

Glucose level ↓ [61]

B6-responsive epilepsies

Pyridoxal and pyridoxal 5′-phosphate ↓ [65-68]

Pyridoxine-dependent epilepsy

Piperideine-6-carboxylate, aminoadipate semialdehyde, pipecolic acid ↑ [67, 68]

The Krebs cycle metabolites (citrate, malate, fumarate, isocitrate, lactate) ↓ [69]

γ-Aminobutyric acid (GABA) pathway metabolites (γ-hydroxybutyrate,

glutamine, glutamate, total GABA, succinic semialdehyde)

↓ [69]

Activity of electron transport chain enzymes ↓ [69]

Pyridoxamine 5′-phosphate oxidase (PNPO) deficiency

Pyridoxamine 5′-phosphate, pyridoxine 5′-phosphate ↑ [65]

Glycine, glutamate and GABA ↓ [65]

Lysine, arginine, tryptophan, methionine, phenylalanine, and threonine ↑ [65]

Pyridoxal 5′-Phosphate (PLP)-Binding Protein (PLPBP) deficiency

Lysine, threonine, asparagine, glutamate, glutamine, proline, alanine,

α-aminobutyric acid, GABA

↓ [66]

Methionine, cystathionine, isoleucine, tyrosine, β-alanine, phenylalanine,

aminoisobutyric acid, tryptophan

↑ [66]

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BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Table 3 (cont.)

Models, biomarkers Responses References

Parkinson’s disease

Dehydrogenase-dependent resazurin metabolism ↓ [70]

Maximal mitochondrial respiration ↓ [71]

Parkin, pink1, and dj-1 protein content ↓ [72]

Lipid peroxidation ↑ [73, 74]

Glutathione ↓ [73, 75]

Superoxide dismutase activity ↓ [73, 74]

Glutathione-S-transferase and catalase activity ↓ [73, 74]

in the whole-body quantitative content of a number of

metabolites (e.g., alanine, taurine, adenosine, creatine,

lactate, acetate, leucine/isoleucine, and histidine). Im-

portantly, content of some metabolites seemed to be

influenced not only by the netting stress exposure

per se, but also by behavioral testing procedures –

which themselves are well-established stressors– indi-

cating the sensitivity of metabolomic characterization

to biochemical alterations induced by altered envi-

ronment [52]. Another proteomic study has demon-

strated that chronic stress (electric shock) upregulates

the whole-body content of several zebrafish proteins

(e.g., HADHB, hspa8, hspa5, actb1, mych4, atp2a1,

zgc:86709, and zgc:86725) regardless of stress predict-

ability [53]. Besides the heat shock proteins, these pro-

tein biomarkers include enzymes, cation transporters,

cytoskeleton proteins, increased level of which may be

considered to play a role in counteracting conditions

associated with elevated fear/anxiety levels.

Moreover, in addition to CNS, cardiovascular sys-

tem is also highly susceptible to the destructive effects

of stress [93, 94]. Proteomic analyses in the chronic

unpredictable stress (CUS) zebrafish model revealed

changes caused by stress in the zebrafish hearts [95].

For example, the observed anxiety-like behavioral

phenotype was accompanied by significantly altered

synthesis rates of several heart proteins involved in

such processes as glycolysis, gluconeogenesis, and hy-

poxia response. Notably, even acute low-level stress af-

fects zebrafish heart proteome, causing decrease in the

rates of degradation of cardiac muscle proteins [54].

Niemann–Pick type C disease. The Niemann–Pick

type C disease (NPC) is a severe autosomal recessive

disease caused by mutations in the NPC1 and NPC2

genes that result in abnormal lysosomal trafficking of

cholesterol and some other lipids [96-98]. This disease

is characterized by a wide range of progressive neu-

rological symptoms including neurodegeneration [99,

100]. Recently, several genetic zebrafish models of NPC

have been successfully developed targeting both NPC1

[55, 56, 59] and NPC2 [57,  58] orthologs of the respec-

tive human genes. Besides the characteristic NPC-like

neurological symptoms and visceral organs damage,

these fish models recapitulate the key biochemical

hallmark of the disease – intracellular accumulation

of unesterified cholesterol. Importantly, free cholester-

ol accumulates in the cells of almost all body tissues

of zebrafish and its presence can be determined by

simple staining techniques [55, 56, 59]. Treatment of

larval zebrafish with 2HPβCD (a drug effective in clin-

ical trials) significantly reduces the cholesterol-posi-

tive staining in neuromast cells [59]. Collectively, this

supports utility of unesterified cholesterol as a suit-

able biomarker of NPC in zebrafish, potentially appli-

cable for high-throughput drug screening. However,

cholesterol is not the only lipid dysregulated in NPC;

altered whole-body distribution of several sphingo-

lipids and phospholipids in the npc1

–/–

zebrafish has

been revealed in a recent study using elaborated 3D

MALDI mass spectrometry imaging (MALDI-MSI) spec-

trometry imaging [60]. Thus, unique lipid biosignatures

for different organs could shed light on the nature of

multifaceted pathological manifestations of NPC.

Epilepsy. Epilepsy is a group of severely debili-

tating neurological disorders characterized by sponta-

neous and recurrent seizures. Zebrafish is becoming

increasingly popular as an alternative model organism

in epilepsy research, with multiple pharmacological

(using proconvulsant drugs) and genetic (based on epi-

lepsy-causing mutations) models of epilepsy in zebraf-

ish being developed (for comprehensive review see

[101]). While most of these models employ behavioral

(seizure-like behavior) and electroencephalographic

(EEG) biomarkers, some studies indicate systemic bio-

ILYIN et al.382

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

chemical/bioenergetic dysregulation that accompanies

the observed epilepsy phenotype.

Indeed, the use of biochemical biomarkers is com-

mon in zebrafish models of B6-responsive epilepsies–

a group of rare genetic disorders, characterized by

severe neonatal seizures that can be alleviated by vi-

tamin B6 [66, 102, 103]. For example, aldh7a1

–/–

zebraf-

ish larvae represent the first animal model of pyridox-

ine-dependent epilepsy, as they readily recapitulate the

main biochemical markers of this disorder: accumula-

tion of piperideine-6-carboxylate, aminoadipate semi-

aldehyde (lysine catabolism metabolites, the substrates

of ALDH7a1 enzyme) and reduced levels of B6 vitam-

ers– pyridoxal(PL), pyridoxal 5′-phosphate (PLP), and

pyridoxamine 5′-phosphate (PMP) [67, 68]. Additionally,

these fish exhibit an approximately twice lower sys-

temic levels of γ-aminobutyric acid (GABA) [67]. While

B6 deficiency in the pyridoxine-dependent epilepsy is

known to be caused by chemical complexation of PLP

with piperideine-6-carboxylate [104], the proposed ex-

planation of reduced GABA levels suggests impaired ac-

tivity of PLP-dependent glutamate decarboxylase – the

key enzyme of GABA biosynthesis [67]. A broad spec-

trum of biochemical dysregulations in aldh7a1

–/–

ze-

brafish likely involves reduced levels of tricarboxylic

acid (Krebs) cycle metabolites, GABA pathway metab-

olites, and activity of the electron transport chain en-

zymes [69].

Biochemical biomarkers have also been extensively

characterized in the zebrafish model of pyridoxamine

5′-phosphate oxidase (PNPO) deficiency – a B6-respon-

sive epilepsy caused by mutation in the gene encoding

the PLP biosynthesis enzyme [105]. The UPLC-MS/MS

analyses of extracts of homogenized bodies of pnpo

–/–

zebrafish reveal the accumulation of pyridoxamine

5′-phosphate, pyridoxine 5′-phosphate (substrates of

PNPO), and reduced levels of pyridoxal 5′-phosphate

and pyridoxal (PNPO downstream products) [65].

Moreover, the pnpo

–/–

zebrafish display reduced levels

of glycine, glutamate, and GABA, and elevated levels

of several essential amino acids (lysine, arginine, tryp-

tophan, methionine, phenylalanine, and threonine),

suggesting impaired activity of the PLP-dependent en-

zymes involved in their metabolism [65]. In contrast,

the PLP treatment normalizes aberrant behavior of

pnpo

–/–

zebrafish, increases fish survival, and restores

the levels of glycine, glutamate, and GABA, although

accumulation of pyridoxamine (PM), PMP and essen-

tial amino acids persisted [65, 106].

The PLP-binding protein (PLPBP) deficiency is an-

other B6-responsive epileptic disorder that has been

successfully modeled in zebrafish. The exact function

of PLPBP and etiology of this recently described disor-

der remain unclear [107]. Similarly to human patients,

plpbp

–/–

zebrafish mutants display reduced systemic

concentrations of PLP, pyridoxal, lysine, threonine,

asparagine, glutamate, glutamine, proline, alanine,

α-aminobutyric acid, GABA, and increased concentra-

tions of methionine, cystathionine, isoleucine, tyro-

sine, β-alanine, phenylalanine, aminoisobutyric acid,

and tryptophan [66]. Overall, zebrafish genetic models

of B6-responsive epilepsies display a wide range of bio-

chemical alterations, most of which can be explained

by impaired activity of the B6-dependent enzymes.

These biochemical markers support the ability of these

models to accurately recapitulate human diseases and

can serve as easily accessible tools for the model val-

idation. Moreover, these findings provide valuable in-

sights into pathophysiology of the B6-responsive epi-

lepsies, corroborating, for example, low GABA levels

as a possible mechanism of epileptogenesis.

Mounting evidence supports the link between hu-

man epilepsy and multiple metabolic defects, including

changes in glucose metabolism, mitochondrial respira-

tion, and glycolysis [108, 109]. Metabolism is also dys-

regulated in the zebrafish models of epilepsy [61-64].

For example, in a zebrafish model of Dravet syndrome

(epilepsy caused by mutations in the sodium NaV

channel), typical behavioral and EEG changes are ac-

companied by the decreased rates of mitochondrial

respiration and glycolysis, measured as oxygen con-

sumption rate and extracellular acidification rate, re-

spectively [63]. Further analyses reveal downregulated

glycolytic enzymes and unaltered activity of the elec-

tron transport chain enzymes, suggesting the causal

role of glycolysis dysregulation in this CNS pathogene-

sis [63]. Reduced glycolysis and mitochondrial respira-

tion have also been reported in the zebrafish epilepsy

model caused by STXBP1 deficiency [62], representing

a simple non-invasive metabolism-based approach

that can be used for real-time monitoring epilepsy and

drug responses in zebrafish.

Such strategies may also be useful for uncovering

novel anti-seizure drugs [110], for instance, identifying

vorinostat (a histone deacetylase inhibitor) as a po-

tential anti-seizure compound based on its ability to

restore metabolic parameters (e.g., basal respiration,

maximal respiration, mitochondrial respiration, proton

leaks, and ATP-linked respiration) in both pharmaco-

logical (pentylenetetrazol-induced) and genetic (kcna1-

morpholino-induced knockdown) zebrafish models of

epilepsy [64]. The efficacy of vorinostat as an antie-

pileptic agent has been further validated in mice us-

ing behavioral and EEG markers [64]. Metabolic-based

small molecule screening has also been successful in

the zebrafish model of the Dravet syndrome discussed

above, since PK11195 (a compound known to enhance

gluconeogenesis) normalizes behavior and restores hy-

pometabolic phenotype of mutant zebrafish, correcting

hypoglycemia and reduced rates of mitochondrial res-

piration and glycolysis [61]. In addition to emphasiz-

ing the value of peripheral metabolic biomarkers for

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BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

specific CNS disorders, these findings support the use

of the energy-producing pathways as novel therapeu-

tic targets for the treatment of brain disorders.

Parkinson’s disease. Mitochondrial dysfunctions

and oxidative stress are strongly implicated in the

Parkinson’s disease pathogenesis [111,  112], a highly

prevalent and severely debilitating progressive neu-

rodegenerative disorder. Some drugs that directly in-

terfere with mitochondrial function can induce Par-

kinsonism in both humans and animals [113-115].

Likewise, zebrafish exposed to mitochondrial dehy-

drogenase inhibitors, such as rotenone, 1-methyl- 4-

phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-

4- phenylpyridinium (MPP

), 6-hydroxidopamine and

paraquat, develop motor deficits, loss of dopaminergic

neurons, and γ-synuclein aggregation, providing good

pharmacological models of Parkinson’s disease [116].

Altered mitochondrial function and redox status in

these models are reflected in various biochemical bio-

markers that can be reliably measured in whole-body

zebrafish samples.

For example, MPTP induces mitochondrial dys-

function in zebrafish, as reflected by decreased activ-

ity of mitochondrial complexI and downregulation of

mitochondrial proteins parkin, DJ-1, and PINK1 [72].

These proteins are crucial for mitochondrial homeo-

stasis and oxidative stress protection, and their dys-

function is associated with familial cases of Parkin-

son’s disease [117]. Interestingly, melatonin treatment

restores both proteins content and complexI activity,

as well as normalizes larvae behavior, supporting its

therapeutic potential for Parkinson’s disease [72]. The

whole-larvae mitochondrial bioenergetics is also af-

fected in the paraquat- [71] and MPP

-induced zebraf-

ish models of Parkinson’s disease [70].

Furthermore, the neurotoxin-exposed zebrafish

present classical biomarkers of oxidative stress, such

as increased lipid peroxidation and decreased gluta-

thione level [73-75], including altered activity of the

enzymes participating in the oxidative stress response,

such as catalase [74, 118], glutathione-S-transferase [73,

74, 118], and superoxide dismutase [73,  74]. Notably,

oxidative stress biomarkers are specifically altered in

the intestines and brains of the rotenone-exposed ze-

brafish [73, 74]. Given some evidence of the causal role

of gastrointestinal dysfunction in Parkinson’s disease

[119, 120], the rotenone-exposed zebrafish may rep-

resent an interesting model to study oxidative stress

related gut-brain interactions relevant to Parkinson

pathogenesis [74].

DISCUSSION

As animal models continue to facilitate studying

mechanisms of complex brain disorders and drug dis-

covery, the zebrafish is rapidly becoming a valuable

model object in the translational neuroscience and

probing a growing number of CNS diseases. These fish

share some practical advantages over mammalian

models together with conserved biochemical and neu-

robehavioral features. Overall, like in humans, CNS

pathogenesis in zebrafish is associated with the dis-

ease-specific biochemical changes, impacting concen-

trations of lipids, carbohydrates, proteins, and other

non-signal molecules, as well as enzymes activities

and rates of metabolic reactions. Importantly, the

signs of biochemical dysregulation can be detected

not only specifically in the brain, but also in peripher-

al tissues and whole bodies of zebrafish. This, togeth-

er with the use of elaborated analytic methods (e.g.,

high performance liquid chromatography-mass spec-

trometry [HPLC-MS]), facilitates biomarker detection

process by making it less time- and labor-consuming.

As discussed above, this strategy has been applied to

a diverse group of zebrafish models of CNS diseases

(e.g., stress-related affective disorders, NPC, epilepsy,

Parkinson’s disease), where the non-brain biochem-

ical biomarkers are used for three major purpos-

es (Table 3).

Firstly, they can be used for the validation of a

particular model, provided that the observed biochem-

ical changes parallel those seen in the clinic. This case

is common in the models of CNS diseases with clearly

established biochemical profile, where, for example,

increased levels of lysine catabolites serve as reliable

biomarkers of pyridoxine-dependent epilepsy in ze-

brafish [67, 68]. Another application is drug screening,

where the reversal of the impaired biochemical pro-

file provides a clear indication of drug effectiveness.

Several studies, as mentioned above, show sensitivity

of zebrafish biochemical biomarkers to pharmacolog-

ical interventions [59, 61, 65, 73, 106], thus promoting

the biomarker-based high-throughput drug screening

in zebrafish. Indeed, at least one compound identified

in zebrafish by its ability to restore metabolic aberra-

tions in epilepsy, vorinostat, now undergoes clinical

trials [64, 121]. Finally, zebrafish may serve as a useful

low-cost platform for the development of biomarkers

that provide new insights into the pathophysiology of

CNS diseases, such as the developed zebrafish 3D maps

of lipid distribution in NPC [60] and profiling amino

acid dysregulation in the models of B6-responsive epi-

lepsies [65, 66,  69].

PROBLEMS, LIMITATIONS,

AND FUTURE RESEARCH

Although focused on peripheral biomarkers, only

few studies have actually investigated the samples of

peripheral tissues without brain tissue, rather than

ILYIN et al.384

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

using whole-body samples. As such, it is not always

clear whether the described biomarkers are actually

of peripheral, central, or rather of “systemic” nature.

A major advantage of the whole-body approach is that

it is fast and does not require tissue dissection, hence,

markedly facilitating rapid biomarkers determination.

This is particularly relevant for the experiments with

small-sized larval and embryonic zebrafish. For exam-

ple, in the zebrafish model of the Dravet syndrome, the

low glucose levels clearly correlate with seizure-like

behavior, thus serving as likely biomarkers of drug re-

sponsiveness as well [61]. At the same time, because

such analyses are performed on the pooled homoge-

nized bodies of zebrafish larvae, it is unclear wheth-

er hypoglycemia is due to the increased brain energy

demands or peripheral glucose depletion. On the one

hand, practically speaking, it is not always necessary to

know the exact origin of the biomarker, if it fulfills its

purpose. On the other hand, further studies may need

to use more specific techniques for tissue collection, re-

vealing the exact origin for each biomarker used.

Studying biomarkers in animal models also has

another benefit – the possibility of finding artifacts

specific to the animal model(s), rather than to the mod-

eled disease itself. In the present context, this can be

attributed to distinct biochemical features of zebrafish

(from those seen in mammals), differences in the ap-

plied experimental manipulations that may only mimic

certain, but not all, etiological factors of ‘real’ human

CNS diseases. For example, in the stress-evoked me-

tabolomic responses, the observed biochemical bio-

markers may reflect zebrafish response to specific

experimental conditions (e.g., stressors) rather than

universal reaction to stress per se [52]. Likewise, vari-

ous oxidative stress biomarkers found in the intestines

of zebrafish exposed to rotenone [73, 74]– a compound

known to induce oxidative stress – can simply con-

firm the peripheral activity of the applied neurotoxin,

rather than inform us on Parkinson’s pathogenesis.

To overcome this problem, one strategy can be to test

such findings using different animal models, also in-

creasing the range of disease-inducing factors (i.e.,

combining pharmacological, genetic, and environmen-

tal manipulations) and employing a wider range of

model organisms, aiming at a greater generality of a

model. Translational relevance of the specific disease

biomarker in question can then be more reliably estab-

lished based on how it integrates into the existing com-

plex pathophysiological picture of the modeled disease.

Finally, the zebrafish as a model organism has

some other, species-specific limitations (Table 1) that

may become critical for CNS disease modeling. Forin-

stance, the zebrafish possesses several neuroanatom-

ical features (e.g., the lack of neocortex) that, albeit

characteristic of all lower vertebrates [16], can hin-

der direct translation of zebrafish disease phenotypes

into humans [122]. Another example is the lack of

midbrain dopaminergic neurons in zebrafish, which

is particularly pertinent when modeling Parkinson’s

disease. In humans, Parkinson’s disease is character-

ized by degeneration of dopaminergic neurons in the

substantia nigra and ventral tegmental area situated

in the midbrain [123, 124]. However, in the zebrafish

models, it is the diencephalic populations that are

primarily affected [113, 125,  126]. While some studies

propose that dopaminergic neurons in the dienceph-

alon of zebrafish may serve as functional analogs of

the human midbrain neuronal populations, this ques-

tion is debated [38, 127,  128]. Thus, while behavioral

and physiological phenotypes observed in zebrafish

models of Parkinson’s disease resemble those in mam-

mals, the degree to which the findings from zebraf-

ish studies can be extrapolated to humans remains

uncertain.

Furthermore, due to additional round of genome

duplication that occurred in the teleost fishes millions

of years ago, zebrafish have more copies of many or-

thologous human genes, including those implicated

into CNS diseases [129, 130]. This, in turn, may com-

plicate such research in zebrafish, including devel-

opment of the genetic knock-out models and inter-

pretation of the results of pharmacological studies.

Furthermore, while zebrafish is perfectly suitable for

administration of water-soluble drugs (through water

immersion), some compounds are water insoluble and

may necessitate the use of organic solvents or injec-

tions, thus increasing the number of factors influenc-

ing the experiment [131]. Lastly, being a relatively new

model object in neuroscience, zebrafish lack some con-

venient features of the traditionally utilized rodents,

such as the availability of multiple fully characterized

inbred strains or behavioral tools [26, 132]. Although

many zebrafish behavioral paradigms have been suc-

cessfully adopted from rodent models [83, 133-135],

some complex behaviors (e.g., fine motor coordina-

tion or parental care) cannot be assessed in zebrafish

experiments despite being highly relevant to many

CNS disorders.

CONCLUSION

Brain disorders often have pathological manifesta-

tions occurring peripherally or at the whole-organism

level, which strongly supports the use of peripheral

biomarkers for diagnostic and therapeutic purposes.

Zebrafish models are becoming an important tool in

the field of translational neuroscience [16, 136], also

providing useful models of human CNS pathologies,

where reliable biochemical changes can be detected ei-

ther in samples of peripheral tissues or whole-bodies.

Zebrafish models often demonstrate similar pattern of

BIOCHEMICAL MARKERS OF BRAIN DISORDERS IN ZEBRAFISH 385

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

biochemical dysregulation to those observed in clin-

ical studies, supporting the developing utility of ze-

brafish to study peripheral biochemical biomarkers of

brain disorders, and their value for the mechanistical-

ly-driven, target-based discovery of novel therapies.

Contributions. A.V.K. conceived and supervised

the study; N.P.I., T.O.K., and S.L.Kh. analyzed the data

and discussed findings with input from all authors;

N.P.I. and K.A.D. wrote the manuscript; E.V.P. and A.V.K.

edited the manuscript.

Funding. The work was financially supported by

the St. Petersburg State University budgetary funds

(Project ID 94030626). A.V.K. and T.O.K. are support-

ed by the Sirius University of Science and Technology

budgetary funds (Project ID NRB-RND-2116).

Ethics declarations. This work does not contain

any studies involving human and animal subjects.

The authors of this work declare that they have no

conflicts of interest.

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