Strategies for the Modulation of Mitochondrial Metabolism and Activity in the Treatment of Neurodegenerative Diseases: A Systematic Review

1. Introduction

Due to the abrupt aging of the population in the last decades, neurodegenerative diseases (NDDs) are raising their prevalence. This upcoming situation challenges our society to redouble our research efforts in the search for effective treatments to face these escalating health and societal problem. Not surprisingly, finding new treatments for NDDs stands out as the central focus of many research endeavors in recent times.

The term NDDs copes with a long list of diseases that includes progressive loss of nerve structure or function; containing a broad spectrum of disorders such as amyotrophic lateral sclerosis, multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, multiple system atrophy, tauopathies, and hereditary ataxias, among others.

Linking the pathological mechanisms of these varied diseases, mitochondrial dysfunction has been described in many of them. In the last years, many studies on NDD have focused on this aspect, such as in Parkinson’s disease and in other conditions like and in other conditions like Leber’s hereditary optic neuropathy¹, or autosomal dominant optic atrophy ¹. Thus, the regulation of mitochondrial metabolism and activity have been identified as potentially relevant therapeutic targets for the treatment of NDDs.

The intrinsic characteristics of the nervous system, and the integrative and behavioral outcomes of its dysfunction make almost imperative the use of animal models in the quest for valuable treatments in the study of NDDs. However, the wide use of rodents as animal models for NDDs implies a high time- and economic-cost. As an alternative, zebrafish (Danio rerio) has become an established and increasingly popular animal model for the characterization of NDDs and the search for fruitful treatments to defeat them.

The relevance of the use of this freshwater fish lays down in the fact that 70% of its genome is shared with humans. Additionally, the zebrafish has a central nervous system organization similar to that of mammals, including humans². Moreover, the ease for gene manipulation allows this species to be used to create quickly and efficiently genetic animal models for specific disease variants. Available gene-editing tools, such as CRISPR-Cas9 variants, enable the modification and breeding of animal models to replicate the specific characteristics of the pathology under investigation, such as Alzheimer’s disease³, Parkinson’s disease⁴, among others. In neuroscience, their transparency during the larvae phase allows the visualization of the central nervous system during developmental studies and their social and cognitive abilities identify zebrafish as a good model for behavioral studies.

These useful features together with their small size, making easy to breed them in high numbers, and their quick development, which reduces experimental costs and increases research production, spot out zebrafish as an excellent animal model that could be used to generate good platforms for quick and effective testing of drugs.

Highlighting the importance of finding novel strategies to ameliorate or reverse the clinical course of the broad spectrum of NDDs, this systematic review aims to provide a complete overview of the most recent literature about compounds with high potential to modulate the mitochondrial metabolism and activity that could be tested for therapeutic purposes in the field of NDDs and that have used zebrafish as animal model.

2. Materials and Methods

The reporting of this review was based on the Preferred Reporting Items for Systematic reviews and Meta-analyses statement (PRISMA)⁵.

2.1. Search Strategy

We conducted a comprehensive literature review, utilizing the following databases: Embase, Web of Science, PubMed, and Scopus in December 2024. Articles published in the English language were evaluated using a specific key term. The search query included keywords combined with Boolean search operators, as follows: mitochondrial metabolism AND therapy AND neurodegenerative diseases AND zebrafish (Table 1). Relevant publications from research and reviews articles published before December 20, 2024, were extracted. Two distinct observers, PVG and BGD, performed the literature search independently to identify articles that potentially met the inclusion. Initially studies were screened based on records identified through database searching; duplicate articles were excluded using Mendeley software tool. Subsequently, articles were screened based on the analysis of the title and abstract according to the predefined inclusion and exclusion criteria (Table 2).

If abstracts fit the criteria, a comprehensive full-text analysis was conducted using the same inclusion criteria. The extracted information has been supplemented using the software tools listed in Table 3. The results extracted from the comprehensive analysis of all the articles are shown in Table 4.

2.2. Search and selection of elegible studies

A total of 176 entries were collected from the primary search, using only the databases mentioned above. All references and citations were managed using Mendeley software (Version 2.125.2) to avoid duplication and ensure proper organization ¹². After removing the duplicate entries, a total of 129 articles remained for the screening of title and abstracts. Among these, 23 entries were eliminated based on the inclusion and exclusion criteria. Subsequently, 106 articles were eligible for full-text assessment, of which 26 studies were excluded due unavailability of full text.

Finally, a total of 34 articles were finally included for data extraction and analysis in this review based on the adopted inclusion criteria. The workflow chart for the selection of eligible articles is shown in Figure 1.

3. Results and Discussion

3.1. Molecules as potential regulators of mitochondial metabolism for treating neurodegenerative diseases

In recent times, new treatments proposed to alleviate various NDDs have focused on seeking modulators of mitochondrial metabolism, which is dysfunctional in many of these pathologies¹³. A summary of the compounds characterized with this activity is shown in Table 4.

3.2. Methological Quality Assessment

Animal experimentation remains crucial for evaluating drugs in the context of neurodegenerative diseases. To assess the methodological quality of the studies included in this systematic review, the SYRCLE tool has been employed. The SYRCLE's Risk of Bias (RoB) tool is specifically designed for animal studies. The criteria included are: random allocation sequence; similar baseline characteristics; allocation concealment; random housing; blinded intervention; random selection for outcome assessment; blinded assessment of outcome; incomplete outcome data; selective outcome reporting; other sources of bias. Each included study was assigned a quality score, with a maximum possible score of 10 points.

According to the article by Hooijmans et al. (48), "Yes" indicates a low risk of bias in the discussed aspect, "No" signifies a high risk of bias, and "Unclear (NC)" means the aspect is not sufficiently addressed in the data provided by the study (Table 5).

The quality items score of each study is ranged from one to seven points, with a mean of 3.29 out of a total 10 points. No study declares a proper concealed allocation, and only one (2,94%) states random housing. The risk of bias in these studies due to the lack of randomization might be medium-low acknowledging in 31 (91.17%) very similar baseline characteristics, and proper reporting bias (34, 100%) free of selective outcome reports.

However, there is a main type of high risk of bias among these publications that is the lack of blinding both in caregivers and investigators (only in 5 studies, 14,70%) and in the outcome assessors (only in 9 articles, 26,47%). In addition, few of the publications mention a sample size calculation. The sample size in animal studies should be large enough to detect biologically significant differences, while also being small enough to minimize unnecessary animal sacrifice. Therefore, this lack of information could harm the internal validity of the evidence from these animal studies.

3.3. Analysis of Potential Mitochondrial Regulator Characteristics

In line with the search for drugs that allow regulating mitochondrial metabolism to improve, prevent, or even cure the mentioned pathologies, numerous studies focus on testing compounds of diverse origins. Some compounds could be considered of endogenous origin, as they are naturally produced by the body, such as neuroglobin⁴⁹ and coenzyme Q10 ⁵⁰ (Metabolite (19%) in Figure 2). Others have an exogenous origin, coming, for example, from plant extracts (35%), like naringenin³⁵. As we can see in the Table 1, most of the studies that presented a mitochondrial regulator compound of exogenous origin, i.e. Berberine derivative¹⁷, naringenin³⁵, theacrine⁴⁶, paeonolum³⁹, BmE-PtNPs¹⁹, acteoside¹⁵, hesperidin²⁹, schisantherin A⁴³, quercetin⁴², 24-epibrassinolide²⁵, and 11-dehydrossinulariolide²⁴ have a naturally occurring chemical origin, as they are extracted from various plant species (Figure 2).

The rest of the compounds are grouped into compounds of natural origin from fungi (3%), bacteria (3%), animal (5%), synthetic (14%) or semisynthetic (16%) (Figure 2) used in other pathologies, acetyl-DL-leucine ¹⁴, terazosin ⁴⁵, and trifluoperazine ⁴⁷, and those with no precise mention of their origin in the study (Figure 2) Compounds of fungi³⁶, animal²⁴ and bacterial⁴⁰ origin are the least represented.

3.4. Mitochondrial Regulation Mechanisms Analysis

Regarding the mode of action of the studied compounds, most of them affect molecular mechanisms related to mitochondrial metabolism and activity per se, such as mitochondrial respiratory chain^19,23,46, mitochondrial stress^26,27, mitochondrial cell death³⁹, intrinsic mitochondrial apoptotic pathway¹⁸, ubiquitin-dependent mitophagy or receptor-mediated mitophagy^17,30,35,47, whereas the others also involve the activities of other organelles or cell functions, such as endoplasmic reticulum^37,41, i.e. Ca²⁺ transfer from the endoplasmic reticulum to the cytosol or mitochondria⁴⁴, endoplasmic reticulum stress-induced apoptosis^26,27, endoplasmic reticulum mediated phagocytosis ¹⁴, or cell signalling pathways, such as AMPK and NF-κB¹⁵, p-CREB and Nrf2²⁴, P53⁵¹, iNOS/NO⁴², MAPK, PI3K/AKT and GSK3β⁴³, or HIF-1 signalling pathways⁴⁵ (Table 1; Figure 3).

3.5. Analysis of the Neurodegenerative Disease Models.

Other interesting aspect is the high number of articles that search for therapies against Parkinson’s disease in zebrafish, testing different compounds (22 out 37) (Figure 4). Parkinson’s disease is the second most prevalent neurodegenerative disorder, with progressive depletion of dopaminergic neurons in substantia nigra pars compacta ⁵². The cause of the disease is unknown, but there are some potential risk factors, such as genetic factors⁵³, age⁵⁴ or environmental agents⁵⁵ that must be take into account.

The notable prevalence of Parkinson’s disease, as identified through the systematic search, is an aspect that warrants further analysis. One potential explanation for this could be that genetic models in rodents have not fully replicated the clinical and neurological characteristics of the pathology⁵³, as age-dependence and progressive symptoms. In efforts to generate new disease models, chemicals have often been used as an alternative, which have been described to reproduce the symptoms of the disease⁵⁵.

One of these chemicals compounds is 6-OHDA. This synthetic compound, analogous to norepinephrine and dopamine⁵⁶, is used to induce Parkinsonian symptoms in animal models, such as zebrafish ³⁵. Neurotoxins with the same effect, including MPP+¹⁸, are also found. However, 6-OHDA does not cross the blood-brain barrier, whereas MPP+ does, resulting in different mechanism of action⁵⁷. Zebrafish assays to test drugs against human diseases are becoming popular during recent years^58–61. Among these assays, only a few succeeded in their purpose, being intensively used in drug screenings against osteoporosis^58–61. In these cases, zebrafish assays were found even more useful than rodent assays for specific metabolic reasons, such as osteoporosis inducibility by anti-inflammatory treatments, a peculiar response in humans not found in mice. 6-OHDA or MPP+ could well be narrowing the distances found between Parkinsonian symptoms in humans and rodent models.

5. Conclusions

Overall, this review clearly reveals the prominent position of zebrafish as a perfect animal model to develop novel assays in the search of potential high-throughput drug-screening pipelines against neurodegenerative diseases in humans.

Neurodegenerative diseases are currently difficult to study, and in many cases, truly effective drugs have yet to be found. This has led to the search for the new compounds that can act on the molecular targets affected in the pathology under study. Given the difficulty in clarifying the causes of the disease and the need to generate animal models that replicate human symptomatology, the use of zebrafish as the preferred model for screening potential drugs is well justified. Drugs with anti-inflammatory, antioxidant, and other activities related to the molecular mechanisms affected in the pathology.

Rodent models, which have also been used historically, sometimes fail to faithfully reproduce disease symptomatology in cases such as Parkinson’s disease. For these reasons, zebrafish is emerging as an alternative, valuable model for screening new potential drugs.