The snake-venom-nerve growth factor-inspired custom peptide downregulates two novel miRNAs (cel-miR-8207-3p and cel-miR-57-3p) for neuroprotection against paraquat-induced toxicity in Caenorhabditis elegans

doi:10.21203/rs.3.rs-4847479/v1

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The snake-venom-nerve growth factor-inspired custom peptide downregulates two novel miRNAs (cel-miR-8207-3p and cel-miR-57-3p) for neuroprotection against paraquat-induced toxicity in Caenorhabditis elegans

https://doi.org/10.21203/rs.3.rs-4847479/v1

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Parkinson's disease (PD) represents a significant global health challenge influenced by environmental factors and the accumulation of alpha-synuclein. MicroRNAs (miRNAs), crucial regulators of gene expression, play pivotal roles in neurodegenerative processes. This study is structured into two primary investigations. Firstly, miRNA microarray analysis in Caenorhabditis elegans treated with mouse 2.5 S-NGF and custom peptide (HNP) revealed distinct expression profiles, notably upregulating miR-1-3p and miR-255-3p compared to controls. Pathway analysis implicated these miRNAs in neuroprotective mechanisms, including PI3K, Wnt/β-catenin, and TGF signalling, while downregulated miRNAs were associated with apoptosis, p53, and innate immunity pathways. These results underscore the neuroprotective roles of NGF and HNP through miRNA-mediated mechanisms in C. elegans. Secondly, a comparative analysis of global miRNA profiles between paraquat-induced PD model worms and NGF/HNP-treated worms identified novel miRNAs and revealed inverse expression patterns indicative of miRNA profile restoration by NGF/HNP pre-treatment. Principal component and correlation analyses demonstrated distinct miRNA regulation profiles across treatments. Gene network analysis identified target mRNAs involved in diverse cellular processes, highlighting potential therapeutic roles of NGF and HNP in neurodegenerative diseases like Parkinson's, mainly through regulation of miR-4813-3p associated with α-synuclein clearance. These findings underscore the promise of NGF and HNP as therapeutic strategies targeting neurodegenerative mechanisms mediated by miRNAs.

Custom peptide

Parkinson's disease

microRNA

neuroprotection

paraquat

C. elegans

Neurodegenerative diseases (NDs) such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD) are characterized by neuronal cell death and irreversible loss of cognitive function [1,2]. These diseases feature abnormal protein aggregation, such as amyloid-beta (Aβ) in AD and alpha-synuclein in PD[3].

Research on novel therapeutic molecules to slow or halt the progression of NDs is gaining significant attention [4–6]. Neurotrophins like nerve growth factor (NGF) are vital for neuronal survival and maintenance, and a decrease in neurotrophic factors (NFs) contributes to ND pathogenesis. However, clinical trials using exogenous NFs have failed mainly due to poor pharmacokinetics [7,8].

MicroRNAs (miRNAs) are small non-coding RNAs that regulate various biological functions, including ageing, proliferation, differentiation, apoptosis, inflammation, immune response, and neurodegeneration, by targeting specific genes [9]. Altered miRNA expression is linked to several diseases, including NDs [10,9,11–13].

In 1993, Lee et al. reported the first miRNA, lin-4, discovered in Caenorhabditis elegans (C. elegans), which targets lin-14 and regulates the ageing process in C. elegans [14]. Since then, C. elegans has been used as a model system for researching miRNAs related to neuronal development and their target genes [15–17]. C. elegans is a model system for studying miRNAs related to neuronal development due to its conserved miRNAs, known neural networks, and neuroanatomy [9]. Moreover, it is in high demand to understand the role of miRNAs in neurobiology and their dysfunction related to medical implications.

Exposure to neurotoxic pollutants like paraquat (PQ) increases the risk of PD-like pathology [18]. PQ is a mitochondrial toxicant herbicide used widely in agriculture and is employed in model organisms to study PD pathogenesis and neuroprotective treatments [19,20]. Previous studies have reported the dysregulation of the miRNAs in the different mutant models of C. elegans or toxicants against heavy metals or nanomaterials [21–23]. However, the protective effects of peptides or natural compounds against pesticide toxicity and the influence of neurotrophins or mimetics on global miRNA expression in C. elegans have not been well documented. Considering the advantages, C. elegans was used as a model system for miRNA next-generation sequencing analysis in this study. The neuroprotective properties of two computationally designed custom peptides [(trideca-neuropeptide (TNP) and heptadeca-neuropeptide (HNP)] to prevent PQ-induced toxicity and neurodegeneration in vitro and in vivo models have been demonstrated [24,25]. However, the HNP showed marginally better neuroprotective activity against paraquat in both models.

Therefore, this study explores the role of miRNAs in the neuroprotective effects of the custom peptide HNP in the C. elegans model. We investigated HNP-regulated miRNAs and their potential to induce signalling pathways similar to those regulated by mouse 2.5S-NGF (positive control), highlighting the neurotrophic and neuroprotective mechanisms. This study is the first report on NGF-regulated miRNAs in C. elegans. Given our previous findings on the neuroprotective potential of synthetic custom peptides, we aimed to study the miRNA expression profile involved in neuroprotection against PQ-induced PD in C. elegans.

2.1. Materials and reagents

S Biochem, Thrissur, Kerala, India, synthesized custom peptide HNP (NH2-HGGKGIGKGGTGGAGVG-COOH)]. Mouse 2.5-S-NGF was obtained from Sigma Aldrich, USA. RNeasy Plant Mini Kit was obtained from Qiagen and DNase I (RNAse-free) from New England Biolabs (NEB) (Cat# M0303L kit). All chemicals were of analytical grade from Sigma, Merck, Hi Media, Invitrogen, and Thermo Fisher Scientific USA. C. elegans Bristol wild-type strain N2 strain and the bacterial control food Escherichia coli (E. coli) OP50 (OP50) were purchased from Caenorhabditis Genetics Centre (CGC), University of Minnesota, USA.

2.2. Maintenance of C. elegans strain

Wild-type Bristol strain N2 was used as an in vivo model for determining PQ-induced oxidative stress and its reversal by the custom peptides. C. elegans N2 strains were grown on NGM plates at 20°C in a bio-incubator. Nematode growth medium (NGM) and Escherichia coli strain OP50 were provided as food sources. Synchronization was performed using the standard procedure of the alkaline hypochlorite method described by Riva et al. (2012) with some modifications. The NGM plates with adequate eggs were washed with M9 buffer and collected into a 15 mL conical centrifuge tube. A mixture of sodium hypochlorite (1 mL) and 5 N NaOH (0.5 mL) was added to the 15 mL tube containing worms in 3.5 mL M9 buffer. The tubes were then agitated for 2 min and centrifuged at 7500 rpm at room temperature (RT) to pellet down the eggs. The supernatant was discarded, and the pellet was washed two times with M9 buffer. Then 0.1 mL of M9 buffer was added to the pellet, transferred to the unseeded NGM plate, and incubated at 20°C after adding E. coli OP50 to the NGM plate.

Sequencing of miRNA to study the global miRNA expression profile and comparison between the PQ group and PHNP group of C. elegans

Synchronized L4 stage wild-type N2 strain of C. elegans was subjected to the following treatments at 20°C: (a) 1X PBS treated worms (CT group), (b) 10 mM paraquat treatment for 1 h (PQ group), (c) 50 µg/mL custom peptide (HNP) pre-treatment for 2 h followed by 10 mM paraquat incubation for 1 h (PHNP group), (d) treatment with 50 µg/mL custom peptide HNP for 2 h (HNP group), (e) 50 µg/mL mouse 2.5 S-NGF pre-treatment for 2 h followed by 10 mM paraquat incubation for 1 h (PNGF group), (f) treatment with 50 µg/mL mouse 2.5 S-NGF for 2 h (NGF group). Each treatment group initially contained 250–300 worms, with a total of approximately 1500–1800 worms used in the study.

Total RNA Extraction: Total RNA extraction was performed using the RNeasy Plant Mini Kit (Qiagen). Extracted RNA underwent DNase treatment per the manufacturer’s protocol (New England Biolabs). RNA concentration and quality were measured using a Qubit fluorometer, and RNA integrity was analyzed using an Agilent BioAnalyser. Only samples with a RIN value ≥ 7 were used for RNA library preparation.

RNA Library Preparation and Sequencing: Transcriptomic mRNA libraries were prepared using the QIAseq miRNA Library Kit according to the manufacturer’s protocol. Qualified RNA samples were subjected to either poly-A capture or ribodepletion, followed by RNA fragmentation, 3' ligation, 5' ligation, cDNA cleanup, adapter and barcode ligation, size selection, and amplification. The libraries were quantified using Qubit and BioAnalyser (Agilent). Sequencing was performed on an Illumina 150 bp PE platform by Biokart India Pvt Ltd, Bangalore, India.

Data Processing and Analysis: Total reads generated from the libraries underwent quality control using FastQC (v0.11.8), and low-quality reads and adapters were removed using Trim Galore (v0.6.7). De novo transcriptome assembly was conducted using Trinity (v2.13.2). The primary assembly was validated and redundancy removed using CD-HIT and RNA-Seq by Expectation-Maximization (RSEM). Validated transcripts underwent Gene Ontology (GO) analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). Raw read counts were obtained using RSEM (v1.3.3), STAR (v2.7.10a), or Salmon (v1.6.0), and processed using BAM and SAM (v1.14) tools, as well as Picard (v2.18.7). Comparative analysis was performed using the Interactive Gene Expression Analysis Kit for Microarray & RNA-seq Data (iGEAK) (v1.0a), an R (v3.3.2) and JavaScript-based open-source desktop application with the Shiny platform. Differentially expressed genes (DEGs) were identified through Voom/edgeR/limma analysis. MiRTarBase predicted the target genes of miRNAs, and their functions were analyzed via the Panther database. Data generation and analysis were performed at Biokart India Pvt Ltd, Bangalore, India

Statistical Analysis and Normalization of miRNA-Sequencing Data: The fold change of miRNA expression was calculated by averaging the control reads and normalizing each miRNA for individual treatment groups relative to the control before converting to log2 values. miRNAs without reads were excluded from subsequent analyses. Statistical analyses, including normalization (edgeR or voom/limma) and identification of DEGs, were performed using iGEAK (v1.0a), an R and Java-based application. Adjusted P values for differential expression analysis are provided in the supplementary data. Criteria for analysis included a false discovery rate (FDR) threshold of [specify threshold, e.g., 0.05] and a fold change cutoff of [specify cutoff, e.g., 2-fold].

Sample Size Calculation

No formal statistical methods were employed to predetermine the sample size. The number of subjects used in this study was determined based on previous studies of a similar nature, ensuring sufficient statistical power and validity [24,26]. Sample size determination was guided by standard practices in the field to ensure robust and reproducible results.

3.1 Identification of mouse 2.5 S-NGF/custom peptide (HNP)-regulated miRNAs in C. elegans by miRNA microarray analysis

The miRNA microarray analysis unveiled distinct expression patterns of twenty miRNAs in the NGF-treated C. elegans group, as summarized in Table 1a. Specifically, ten miRNAs exhibited upregulation compared to the CT group, while ten were downregulated. Similarly, thirteen miRNAs displayed differential expression in the HNP-treated C. elegans group (Table 1b), with ten miRNAs being upregulated and three miRNAs downregulated relative to the CT group. Notably, several miRNAs including cel-miR-255-3p, cel-miR-1-3p, cel-miR-62, cel-miR-85-3p, cel-miR-71-3p, cel-miR-83-3p, cel-miR-84-5p, and cel-miR-4936 were found to be expressed in both the NGF and HNP treatment groups. Cel-miR-1-3p and cel-miR-255-3p exhibited significant upregulation in both NGF and HNP groups compared to the CT group. Furthermore, Table 1c lists the annotated pathways associated with potential miRNA targets identified in the NGF and HNP treatment groups. These findings underscore the diverse regulatory roles of miRNAs in neurite outgrowth and neuroprotection conferred by NGF and HNP treatments in C. elegans.

The potential miRNA target pathways in the NGF and HNP groups of C. elegans are detailed in Table 1c. The volcano plot (p-values vs. fold change) illustrated the differential miRNA expression between (i) the NGF and CT groups (Fig. 1a) and (ii) the HNP and CT groups (Fig. 1b). Heat map analysis displayed distinct gene expression profiles between the NGF and HNP groups compared to the CT group (Fig. 1c-d). The predicted target genes of the differentially expressed miRNAs were associated with various signalling pathways and cellular processes. Upregulated miRNAs in the NGF and HNP groups were linked to pathways such as PI3K, Wnt/β-catenin, TGF signalling, axon development, transcriptional regulation, and cellular development. Conversely, the target genes of downregulated miRNAs were implicated in apoptotic pathways, p53 pathways, neurological diseases, Ras pathways, and innate immune response pathways in both the NGF and HNP groups. The role of some upregulated miRNAs in different stages of neuronal development in the NGF and HNP groups of C. elegans is depicted in Fig. 2.

A comparison of the differential expression of global miRNA profiling between PQ -treated C. elegans and mouse 2.5 S-NGF and HNP groups of C. elegans

Several newly discovered miRNAs, not previously identified or catalogued in existing miRNA databases (novel miRNA sequences), were identified across all treated groups. The sequences of these novel miRNAs are listed in Supplementary Table S1 (1a, 1b, 1c, 1d, and 1e). The differentially expressed miRNAs and their respective logFC values for the comparisons of the CT group vs PQ group, PNGF group vs PQ group, and PHNP group vs PQ group of C. elegans are detailed in Table 2a and Table 2b. Principal component analysis (PCA) score plots showed clustering patterns: Group 1 consisted of CT, PT, PNGF, and NGF groups of C. elegans (Fig. 3a), while Group 2 included CT, PT, PHNP, and HNP groups (Fig. 3b). The clustering between Group 1 and Group 2 is illustrated in Fig. 3c. Correlation plots, with red indicating positive correlation and blue indicating negative correlation, were constructed for different groups (Fig. 3d-f).

Volcano plots (p-values vs. fold change) displayed the differential expression of miRNAs between (i) PNGF and PQ groups (Fig. 3g) and (ii) PHNP and PQ groups of C. elegans (Fig. 3h). Heat map analysis compared miRNA expression profiles across different treatment groups (Figs. 4a-b). The analysis reveals a reciprocal regulation pattern of miRNAs between the PQ and treated groups. Specifically, miRNAs upregulated in the PQ group (compared to the CT group) exhibit downregulation in the PNGF and PHNP groups. In contrast, downregulated miRNAs in the PQ group show upregulation in the PNGF and PHNP groups. This inverse relationship suggests that the pre-treatments with NGF and HNP modulate the miRNA expression profile towards a state that counteracts the alterations induced by PQ exposure. These findings indicate that the treatments may restore or normalize the miRNA expression disrupted by PT, potentially contributing to their neuroprotective effects. Gene network analysis identified downstream mRNA targets modulating multiple classes of proteins (Supplementary Table S2). The table lists the downstream mRNA targets of miRNAs identified in the PNGF and PHNP groups of C. elegans, categorized according to their Panther protein classes. The predicted targets encompass a diverse range of protein functions essential for various cellular processes, including ion channels, transmembrane signal receptors, RNA metabolism proteins, acyltransferases, apolipoproteins, DNA-directed RNA polymerases, chaperones, peroxidases, and numerous others. These proteins are implicated in critical biological functions such as transcriptional regulation, cellular development, signal transduction, and metabolic pathways. Detailed miRNA and target mRNA networks for the PNGF and PHNP groups of C. elegans are visualized in Figs. 5a and 5b, respectively, highlighting the complexity of regulatory interactions underlying neuroprotective mechanisms in these experimental models.

Since there is no effective therapy or treatment to prevent the progression of NDs such as AD and PD, which have emerged as a significant problem in neurobiology in recent years, thus more research efforts are being made to develop innovative neuroprotective therapeutics [27]. The potential role of miRNA in ND has encouraged the finding of novel therapeutic targets [28,29].

Since neurotrophic factors cannot cross the blood-brain barrier (BBB) and must be administered intracerebrally, their use in treating central nervous system dysfunction is even more complicated. However, small synthetic peptides can be used as an alternative method with several advantages over large protein molecules in treating neuronal disease [30]. Some peptides named MIM-D3 (TrkA agonist) ameliorate corneal injury in rats with dry eye syndrome [31]. Similarly, GK-2 (NGF mimetics) improved neurological disorders such as AD, PD, and brain ischemia without adverse effects [32,33].

In vivo model organisms such as C. elegans provide a valuable platform for elucidating the roles and mechanisms of miRNAs in neuronal development and function [9]. Previous studies have highlighted the involvement of cel-miR-71, cel-miR-84, and cel-miR-1 in various stages of neuronal development [9]. Our investigation observed significant upregulation of these miRNAs in both NGF and HNP-treated groups of C. elegans. These findings underscore the neurorestorative and neurotrophic potential of HNP, which appears comparable in efficacy to mouse 2.5 S-NGF.

Particularly noteworthy were cel-miR-1-3p and cel-miR-255-3p, which exhibited robust expression following treatment with mouse 2.5 S-NGF and custom peptide HNP in C. elegans. Our study further delved into their respective target genes: cel-miR-1-3p targets mef-2, unc-63, unc-29, and unc-38, influencing pathways related to oxidative stress response, p38/MAPK signalling, and nicotinic acetylcholine receptor signalling (RefSeq, miRBase, ENA, WormBase). Similarly, cel-miR-255-3p modulates genes such as kcnl-2, idhb-1, suca-1, pyk-1, and tos-1, thereby impacting pathways involved in G protein signaling, pyruvate metabolism, and integrin signaling (RefSeq, miRBase, ENA, WormBase). These findings contribute to our understanding of how miRNAs regulate key pathways in neuronal processes, potentially offering insights into therapeutic strategies for neurodegenerative disorders.

The p38/MAPK signalling pathways are pivotal in neuronal differentiation, as evidenced by previous research from our lab demonstrating induction by mouse 2.5 S-NGF [34–36]. Derived from the TrkA binding region of snake venom NGF, the custom peptide utilized in this study also promotes neuronal differentiation and growth [24,25]. Our findings indicate that similar to mouse 2.5 S-NGF, HNP upregulates cel-miR-1-3p, modulating p38/MAPK pathways to facilitate neuronal differentiation in C. elegans.

The accumulation of α-synuclein is a hallmark of Parkinson's disease (PD) progression, observed in the PQ -induced PD model [37]. Removal of aggregation of this protein is a required mechanism for improving PD. Studies have identified cel-miR-4813-3p as a regulator of protein homeostasis involved in α-synuclein clearance in neuronal cells [21,22]. Our lab has demonstrated the neuroprotective potential of HNP [24,25], and we found that cel-miR-4813-3p is downregulated in the PQ -treated group of C. elegans, leading to α-synuclein deposition and PD progression. Notably, the upregulation of cel-miR-4813-3p in the PHNP and PNGF groups strongly suggests the therapeutic potential of these peptides through miRNA regulation.

The phosphoinositide 3-kinase (PI3K) pathway promotes longevity and neuronal development and is regulated by cel-miR-236-3p [38]. PQ treatment downregulates cel-miR-236-3p expression, whereas its upregulation in the PHNP and PNGF groups of C. elegans supports the neuroprotective role of HNP via miRNA modulation. Additionally, two novel miRNAs, cel-miR-8207-3p and cel-miR-57-3p, are upregulated by PQ treatment and downregulated by mouse 2.5 S-NGF and HNP pre-treatment. These miRNAs' functions remain unknown and warrant further exploration.

Our study elucidates how HNP influences neuronal differentiation, mitigates α-synuclein aggregation in PD models, and promotes neuroprotection through miRNA-mediated pathways in C. elegans.

In conclusion, this study demonstrates that PQ -induced alterations in miRNA expression in C. elegans correlate with disruptions in developmental processes, longevity regulation, α-synuclein degradation, and oxidative stress responses, which are critical in neurodegenerative disease pathogenesis. The upregulation of miRNAs associated with apoptotic pathways, reduced lifespan, innate immune responses, and metabolic dysregulation further suggests their role in promoting neurodegenerative disease progression. Pre-treatment with mouse 2.5 S-NGF and custom peptide HNP effectively restored the expression of dysregulated miRNAs induced by PQ treatment, highlighting their potential as neuroprotective strategies. These findings underscore the therapeutic promise of targeting miRNA-mediated pathways in neurodegenerative diseases, offering new avenues for developing targeted therapies to mitigate disease progression.

Alzheimer's disease

Aβ

Amyloid-beta

BBB

Blood-brain barrier

C. elegans

Caenorhabditis elegans

Control

DAVID

Database for Annotation

Visualization

and Integrated Discovery

Gene Ontology

Huntington's disease

HNP

Heptadeca-neuropeptide

MAPK

Mitogen-Activated Protein Kinase

miRNAs

MicroRNAs

NDs

Neurodegenerative diseases

NFs

Neurotrophic factors

NGF

Nerve growth factor

NGM

Nematode growth medium

PCA

Principal Component Analysis

Parkinson's disease

PHNP

Paraquat and HNP-treated group

PI3K

Phosphoinositide 3-kinase

PNGF

Paraquat and NGF-treated group

Paraquat

RSEM

RNA-Seq by Expectation-Maximization

Room temperature

TNP

Trideca-neuropeptide.

Conflict of interest:

The authors have no relevant financial or non-financial interests to disclose.

Author Contribution

A.K.M. conceived the idea, A.K.M. and D.M. designed the experiments, D.M. performed the analysis, wrote the manuscript, and prepared the figures. A.K.M. and M.R.K. edited and corrected the manuscript. All authors reviewed the manuscript.

Acknowledgements:

The authors thank the SAIC of IASST for the instrumental facility. DM received SRF from the SERB project. This work was funded by the Science and Engineering Research Board, New Delhi (EMR/2017/001829) and partially contributed from the core research grant of IASST to AKM.

Data Availability

Data is provided within the manuscript and in supplementary information file.

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Table 1a: The miRNA microarray analysis data, indicating the fold changes of differentially regulated miRNAs in C. elegans treated with 50 µg/mL of mouse 2.5 S-NGF (NGF) group for 2 h compared to the control (CT) group of C. elegans, with statistical significance p < 0.05.

Types of miRNA	logFC (CT vs NGF)	t value	p Value
cel-miR-1-3p	2.69	354.70	0.00
cel-miR-255-3p	2.21	4.48	0.01
cel-miR-83-3p	1.93	8.21	0.00
cel-miR-85-3p	1.92	74.28	0.00
cel-miR-59-3p	1.87	206.60	0.00
cel-miR-62	1.72	5.10	0.01
cel-miR-84-5p	1.60	328.20	0.00
cel-miR-794-5p	1.54	9.79	0.00
cel-miR-795-3p	1.51	3.19	0.03
cel-miR-71-3p	1.30	7.72	0.00
cel-miR-4936	-3.08	-146.30	0.00
cel-miR-67-3p	-3.14	-12.97	0.00
cel-miR-41-5p	-3.27	-5.73	0.00
cel-miR-5545-3p	-3.33	-5.08	0.01
cel-miR-8211-5p	-3.79	-5.45	0.00
cel-miR-8204-5p	-3.88	-10.46	0.00
cel-miR-67-5p	-4.12	-5.04	0.01
cel-miR-8205-3p	-4.81	-12.01	0.00
cel-miR-2217b-3p	-5.04	-4.68	0.01
cel-miR-8202-5p	-5.10	-4.23	0.01

Table 1b: The miRNA microarray analysis data showing the fold changes of differentially regulated miRNA in the C. elegans treated with 50 µg/mL of custom peptide (HNP) group for 2 h compared to the control (CT) group of C. elegans, with statistical significance p < 0.05.

Types of miRNA	logFC (CT vs HNP)	t value	p Value
cel-miR-255-3p	3.47	2.39	0.02
cel-miR-87-5p	3.13	2.09	0.04
cel-miR-1-3p	1.87	18.95	0.00
cel-miR-1820-5p	1.54	16.87	0.00
cel-miR-62	1.53	2.11	0.03
cel-miR-358-3p	1.50	2.45	0.01
cel-miR-85-3p	1.38	14.25	0.00
cel-miR-71-3p	1.30	4.91	0.00
cel-miR-83-3p	1.40	11.42	0.00
cel-miR-84-5p	1.55	7.65	0.00
cel-miR-791-3p	-1.36	-7.17	0.00
cel-miR-789-5p	-1.44	-2.55	0.01
cel-miR-4936	-1.55	-13.84	0.00

Table 1c: Pathways annotation of potential miRNA target genes in HNP and mouse 2.5 S-NGF-treated C.elegans. The p-values for both treatment groups are also included. If p-values are not provided, it indicates that those pathways are absent in the particular treated group.

Pathways of potential miRNA target genes	The p-Value of HNP-treated C.elegans	The p-Value of mouse 2.5 S-NGF treated C.elegans
BINDING: ATP		0.463
CARBOHYD: N-linked (GlcNAc) asparagine	0.183	0.239
cel01100: Metabolic pathways		0.000
cel01200: Carbon metabolism	0.006	0.004
cel01230: Biosynthesis of amino acids	0.001	0.000
GO:0000166~nucleotide binding	0.299	0.198
GO:0000287~magnesium ion binding	0.009	0.006
GO:0004888~transmembrane signaling receptor activity	0.255	0.209
GO:0005216~ion channel activity	0.367	0.321
GO:0005230~extracellular ligand-gated ion channel activity	0.249	0.214
GO:0005524~ATP binding	0.425	0.308
GO:0005739~mitochondrion	0.024
GO:0005886~plasma membrane	0.226	0.055
GO:0006811~ion transport	0.055	0.039
GO:0006813~potassium ion transport	0.069	0.060
GO:0006937~regulation of muscle contraction	0.032	0.028
GO:0007165~signal transduction	0.070	0.109
GO:0007268~chemical synaptic transmission	0.348	0.314
KW-0407~Ion channel		0.481
GO:0016301~kinase activity	0.198	0.279
KW-0460~Magnesium		0.207
GO:0016310~phosphorylation	0.186	0.282
GO:0022848~acetylcholine-gated cation-selective channel activity	0.017	0.014
GO:0022857~transmembrane transporter activity	0.278	0.083
GO:0030054~cell junction	0.165	0.119
GO:0030594~neurotransmitter receptor activity	0.286	0.247
GO:0034220~ion transmembrane transport	0.146	0.123
GO:0040011~locomotion	0.039	0.154
GO:0042391~regulation of membrane potential	0.241	0.215
GO:0043005~neuron projection	0.254	0.189
GO:0045211~postsynaptic membrane	0.063	0.048
GO:0046662~regulation of oviposition	0.056	0.049
GO:0050877~neurological system process	0.226	0.201
GO:0055085~transmembrane transport	0.424	0.191
GO:0060078~regulation of postsynaptic membrane potential	0.002	0.001
GO:0060079~excitatory postsynaptic potential	0.021	0.018
GO:0090326~positive regulation of locomotion involved in locomotory behaviour	0.014	0.012
IPR002394: Nicotinic acetylcholine receptor	0.009	0.007
IPR006029: Neurotransmitter-gated ion-channel transmembrane domain	0.118	0.102
IPR006201: Neurotransmitter-gated ion-channel	0.172	0.149
IPR006202: Neurotransmitter-gated ion-channel ligand-binding	0.176	0.154
IPR011701: Major facilitator superfamily	0.157	0.137
IPR018000: Neurotransmitter-gated ion-channel, conserved site	0.116	0.100
KW-0067~ATP-binding	0.138	0.044
KW-0325~Glycoprotein	0.259	0.265
KW-0406~Ion transport	0.089	0.103
KW-0418~Kinase	0.063	0.119
KW-0443~Lipid metabolism	0.485
KW-0472~Membrane	0.244	0.124
KW-0496~Mitochondrion	0.084	0.121
KW-0547~Nucleotide-binding	0.050	0.012
KW-0628~Postsynaptic cell membrane	0.031	0.021
KW-0770~Synapse	0.133	0.094
KW-0808~Transferase	0.132	0.153
KW-0809~Transit peptide	0.078	0.221
KW-0812~Transmembrane	0.066	0.177
KW-0813~Transport	0.419	0.472
KW-0965~Cell junction	0.098	0.060
KW-1003~Cell membrane	0.030	0.009
KW-1015~Disulfide bond	0.233	0.108
KW-1071~Ligand-gated ion channel	0.035	0.030
KW-1133~Transmembrane helix	0.233
NP_BIND: ATP	0.375	0.146
R-CEL-112314~Neurotransmitter receptors and postsynaptic signal transmission	0.294	0.281
R-CEL-112316~Neuronal System	0.419	0.400
R-CEL-181431~Acetylcholine binding and downstream events	0.074	0.070
R-CEL-195721~Signaling by WNT		0.486
R-CEL-212436~Generic Transcription Pathway	0.224	0.425
R-CEL-3858494~Beta-catenin independent WNT signaling		0.143
R-CEL-556833~Metabolism of lipids	0.327
R-CEL-622323~Presynaptic nicotinic acetylcholine receptors	0.011	0.010
R-CEL-622327~Postsynaptic nicotinic acetylcholine receptors	0.074	0.070
R-CEL-629594~Highly calcium permeable postsynaptic nicotinic acetylcholine receptors	0.074	0.070
R-CEL-629597~Highly calcium permeable nicotinic acetylcholine receptors	0.011	0.010
R-CEL-6798695~Neutrophil degranulation		0.493
R-CEL-71387~Metabolism of carbohydrates	0.031	0.112
R-CEL-73857~RNA Polymerase II Transcription	0.418
R-CEL-74160~Gene expression (Transcription)	0.489
R-CEL-8878171~Transcriptional regulation by RUNX1	0.196
TOPO_DOM: Cytoplasmic	0.062	0.096
TOPO_DOM: Extracellular	0.103	0.072
TRANSIT: Mitochondrion	0.059	0.189

Table 2a: The miRNA sequencing analysis data compares the fold changes in the differential expression of miRNA in the different treatment groups. The experimental conditions are described in the text.

miRNA	Log FC (CT vs PT)	logFC (PNGF vs PT)	Function	References
cel-miR-77-5p	-3.17	1.32	Embryogenesis	Abbott, 2012
cel-miR-4813-3p	-3.05	3.65	Degradation of alpha-synuclein	Sarkar et al., 2022
cel-miR-34-3p	-1.95	1.58	Increasing autophagic flux against oxidative stress	Yang et al., 2013
cel-miR-236-3p	-1.39	2.80	Phosphoinositide 3-kinase (PI3K) pathways for cell growth and development (promote longevity)	Lencastre et al., 2010
cel-miR-46-3p	-1.09	1.36	Development	Lau et al., 2001
cel-miR-786-3p	1.39	-1.16	Fatty acid metabolism	Kemp et al., 2012
cel-miR-1820-5p	1.43	-1.54	Innate immune response	Sun et al., 2016
cel-miR-83-3p	1.75	1.35	Loss of miR-83 extends lifespan	Dzakah et al., 2018
cel-miR-795-3p	2.17	-1.44	Dauer formation	Martinez et al., 2008
cel-miR-57-3p	3.23	-2.38
cel-miR-8207-3p	4.31	-7.62

Table 2b: The miRNA sequencing analysis data compares the fold changes in the differential expression of miRNA in the different treatment groups. For comparison of differential expression of the miRNA, the C.elegans were subjected to the following treatments at 20°C: (a) 1X PBS (control) treated C.elegans (CT), (b) 10 mM paraquat (PT) treatment for 1 h, (c) pre-treatment with 50 µg/mL custom peptide for 2 h followed by 10 mM paraquat treatment (PHNP) for 1 h.

miRNA	logFC (CT vs PT)	logFC (PHNP vs PT)	Function	References
cel-miR-77-5p	-3.17	1.18	Embryogenesis	Abbott, 2012
cel-miR-62	-3.00	3.82	Regulate stress response and increase the lifespan	Lencastre et al., 2010
cel-miR-4813-3p	-3.05	1.20	Degradation of alpha-synuclein	Sarkar et al., 2022
cel-miR-4936	-2.59	1.57	Activation of heat shock factor-1 (HSF-1) to protect the cell from organelle dysfunction	Schreiner, 2019
cel-miR-236-3p	-1.39	1.77	Phosphoinositide 3-kinase (PI3K) pathways for cell growth and development (promote longevity)	Lencastre et al., 2010
cel-miR-83-3p	1.75	1.03	Loss of miR-83 extends lifespan.	Dzakah et al., 2018
cel-miR-57-3p	3.23	-3.23
cel-miR-8207-3p	4.31	-6.03
cel-miR-354-5p	4.60	-5.24	The transforming growth factor beta (TGF -β) signalling pathway shows a protective response.	Wang et al., 2020

No competing interests reported.

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The snake-venom-nerve growth factor-inspired custom peptide downregulates two novel miRNAs (cel-miR-8207-3p and cel-miR-57-3p) for neuroprotection against paraquat-induced toxicity in Caenorhabditis elegans

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Abstract

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1. Introduction

2. Materials and Methods

2.1. Materials and reagents

2.2. Maintenance of C. elegans strain

3. Results

3.1 Identification of mouse 2.5 S-NGF/custom peptide (HNP)-regulated miRNAs in C. elegans by miRNA microarray analysis

4. Discussion

5. Conclusion

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