
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

72666
10.1038/s41598-024-72666-8
Article
Downregulation of Lnc-ABCA12-3 modulates UBQLN1 expression and protein homeostasis pathways in amyotrophic lateral sclerosis
Yu Yujiao
Pang Dejiang
Huang Jingxuan
Li Chunyu
Cui Yiyuan
http://orcid.org/0000-0003-0947-1151
Shang Huifang hfshang2002@126.com

https://ror.org/011ashp19 grid.13291.38 0000 0001 0807 1581 Department of Neurology, Laboratory of Neurodegenerative Disorders, West China Hospital, National Clinical Research Center for Geriatrics, Sichuan University, No.37, Guoxue Lane, Chengdu, 610041 China
13 9 2024
13 9 2024
2024
14 213831 2 2024
9 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron degeneration. Dysregulation of long non-coding RNAs (lncRNAs) has been implicated in ALS pathogenesis but their roles remain unclear. Previous studies found lnc-ABCA12-3 was downregulated in ALS patients. We aim to characterize the expression and function of lnc-ABCA12-3 in ALS and explore its mechanisms of action. Lnc-ABCA12-3 expression was analyzed in PBMCs from ALS patients and correlated with clinical outcomes. Effect of modulating lnc-ABCA12-3 expression was assessed in cell models using assays of apoptosis, protein homeostasis and pathway analysis. RNA pull-down and interaction studies were performed to identify lnc-ABCA12-3 binding partners. Lnc-ABCA12-3 was downregulated in ALS patients, correlating with faster progression and shorter survival. Overexpression of lnc-ABAC12-3 conferred protection against oxidative stress-induced apoptosis, while knockdown lnc-ABCA12-3 enhanced cell death. Lnc-ABCA12-3 maintained protein quality control pathways, including ubiquitination, autophagy and stress granule formation, by regulating the ubiquitin shuttle protein UBQLN1. This study identified lnc-ABCA12-3 as a novel regulatory lncRNA implicated in ALS pathogenesis by modulating cellular survival and stress responses through interactions with UBQLN1, influencing disease progression. Lnc-ABCA12-3 may influence ALS through regulating protein homeostasis pathways.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72666-8.

Subject terms

Non-coding RNAs
Mechanisms of disease
The Sichuan Science and Technology Program2022NSFSC0750 http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 81871000 issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Amyotrophic Lateral Sclerosis (ALS), a progressive neurodegenerative disorder impacting both upper and lower motor neurons, is a prevalent and lethal form of motor neuron disorders (MND) characterized by divergent presentations, rapid progression, and high mortality1,2. ALS is a devastating disease that leads to progressive muscle weakness, loss of motor neurons, respiratory failure, and ultimately, death3. Beyond muscle weakness, some patients may also exhibit cognitive or behavioral changes. The incidence rate of ALS is approximately 0.3 to 2.5 new cases per 100,000 people each year2,4–6. Intriguingly, less than 10% of ALS cases are inherited, termed familial ALS, leaving the vast majority, over 90%, as sporadic ALS (SALS).

Research shows that as much as 98% of the human genome contains non-protein-coding sequences7. Among these, noncoding RNAs (ncRNAs), which lack an open reading frame, and thus, cannot code proteins, have been associated with various cellular regulations and disease progression due to advances in biotechnology. In recent times, studies have highlighted the critical role of defective RNA metabolism contributes to neurodegenerative diseases like ALS through dysregulated mRNAs and ncRNAs, with several ALS causative genes such as TARDBP, FUS/TLS, ANG, and C9ORF72 identified through molecular genetic investigations8,9. Meanwhile, the identification of long non-coding RNAs (lncRNAs) has unveiled new perspectives into the regulation of disease-associated genes, as lncRNAs are known to interact with RNA, DNA, or proteins to either enhance or inhibit the expression of protein-coding genes10. Interestingly, lncRNAs have been implicated in neuronal differentiation, synaptogenesis, and other functional maintenance processes11.

As such, dysregulation of lncRNA expression can play pivotal roles in neurodegenerative diseases. Several lncRNAs have shown dysregulated states in neurodegenerative diseases such as Alzheimer’s, Parkinson’s disease or ALS, with their associated biochemical functions and protein or mRNA regulatory functions being reported12–15. Recent research has provided initial insights into the functions of lncRNAs related to disease pathogenesis. Several studies have investigated specific lncRNAs implicated in ALS and delved into their roles at the molecular level. The nuclear lncRNA NEAT1, consisting of isoforms NEAT1_1 and NEAT1_2, plays a structural role in paraspeckles, with NEAT1_2 being essential for their formation; mutations in ALS-linked RBPs like TDP-43 and FUS that interact with NEAT1 dysregulate its expression levels and paraspeckle formation, potentially contributing to ALS pathogenesis, though more research is needed to clarify these relationships16,17. The expanded GGGGCC hexanucleotide repeat in the C9ORF72 gene represents the most common known genetic cause of both ALS and FTD in many populations, with transcripts of both the sense (C9ORF72-S) and complementary antisense (C9ORF72-AS) sequences harboring the expansion capable of translating into dipeptide aggregates and forming nuclear foci that may contribute to pathogenesis through multiple toxic gain-of-function mechanisms18–20.

Our previous work uncovered potential roles for lncRNAs in ALS by analyzing their expression patterns in peripheral blood leukocytes from ALS patients. Specifically, we found that several lncRNAs, including lnc-ABCA12-3, had reduced expression in peripheral blood leukocytes from ALS patients compared to controls21. Lnc-ABCA12-3 is not a novel lncRNA first identified in the diseases. Prior research has begun to elucidate molecular functions for lnc-ABCA12-3 in other diseases. For example, some studies showed lnc-ABCA12-3 can promote processes like cell migration, invasion and proliferation in esophageal squamous cell carcinoma22. This provides a framework for investigating how dysregulation of lnc-ABCA12-3 expression in ALS, as our previous work revealed, may influence molecular pathways and phenotypes relevant to motor neuron degeneration. Ongoing investigations are exploring both relative to the disease progression and the mechanisms by which lnc-ABCA12-3 specifically contributes to ALS pathogenesis. We discovered that lnc-ABCA12-3 interacts with the ubiquitin-like protein UBQLN1, which plays an important role in maintaining protein homeostasis through pathways such as the ubiquitin-proteasome system and autophagy, thereby contributing to protein homeostasis. Knocking down lnc-ABCA12-3 decreased both the mRNA and protein levels of UBQLN1 so that lnc-ABCA12-3 may participate in regulating cellular protein quality control processes relevant to ALS pathogenesis by associating with and modulating UBQLN1. Our findings help elucidate a potential mechanism whereby dysregulation of lnc-ABCA12-3 contributes to ALS pathogenesis—through influencing UBQLN1-mediated proteostasis networks critical for motor neuron survival and function.

Results

1. Alterations in Lnc-ABCA12-3 expression correlate with disease progression and survival status in SALS patients.

In our previous study, we found that lnc-ABCA12-3 expression was significantly decreased in peripheral blood leukocytes of ALS patients within one year of symptom onset. We hypothesized that alterations in lnc-ABCA12-3 levels may correlate with disease progression dynamics.

To test this, we analyzed clinical data from those patients and measured correlations between lnc-ABCA12-3 expression and ALS Functional Rating Scale (ALSFRS-R) scores/progression rates. We found no correlation with initial ALSFRS-R scores (Fig. 1A). However, disease progression rates calculated from later ALSFRS-R evaluations showed a significant negative correlation with lnc-ABCA12-3 levels, and this relationship strengthened over time (Fig. 1B). We then divided patients into high vs. low lnc-ABCA12-3 expression groups and calculated survival probabilities. The group with higher lnc-ABCA12-3 expression demonstrated improved survival probabilities (P = 0.056), retaining motor function for longer periods before reaching proportional death thresholds (Fig. 1C). These data suggest lower peripheral lnc-ABCA12-3 expression is associated with more rapid disease progression rates and shorter survival times following diagnosis. This supports the hypothesis that dynamic changes in lnc-ABCA12-3 levels correlate with and may influence ALS progression dynamics over time.Fig. 1 Correlation between lnc-ABCA12-3 expression and clinical patient manifestation. (A) Pearson correlation analysis of lnc-ABCA12-3 expression and ALSFRS-R score. (B) Lnc-HIBADH-4 expression in sALS patients with disease progression. (C) Survival analysis of sALS patients with relatively low and high expression of lnc-HIBADH-4 (Kaplan-Meier estimation and the log-rank test).

2. The expressed reduction of lnc-ABCA12-3 promotes apoptosis.

Given our finding that decreased lnc-ABCA12-3 expression correlates with worse patient outcomes, we sought to understand the molecular mechanisms involved at the cellular level. Understanding how lnc-ABCA12-3 impacts cellular physiology and biochemistry could provide insight into its role in ALS pathogenesis. As a first step, we examined whether altering lnc-ABCA12-3 expression affects cell survival in response to oxidative stress. We silenced and overexpressed lnc-ABCA12-3 in cell lines for checking the transfection efficiency (supplemental Fig. 1A-1B). Lnc-ABCA12-3 siRNA3 (referred to as “siRNA” in figures) and lnc-ABCA12-3 overexpression plasmid (referred to as “OE” in figures) were selected based on their sufficiently low or high endogenous expression levels, respectively. Flow cytometry analysis revealed a higher proportion of apoptotic cells in lnc-ABCA12-3 silenced conditions compared to controls with an oxidizing agent treatment (Fig. 2A). Similarly, Hoechst staining showed more fluorescent spots indicating apoptotic cells in silenced samples (Fig. 2C). Western blotting for cleaved-PARP, a marker of apoptosis, exhibited the same trend (Fig. 2D). Conversely, lnc-ABCA12-3 overexpression led to a lower proportion of apoptotic cells versus controls based on all three assays (Fig. 2B, C and E). These data demonstrate that reducing lnc-ABCA12-3 expression enhances cellular susceptibility to oxidative stress-induced apoptosis, whereas overexpressing lnc-ABCA12-3 confers a protective effect against apoptosis. This provides a first step toward elucidating how lnc-ABCA12-3 impacts molecular mechanisms relevant to ALS pathogenesis.

3. Lnc-ABCA12-3 binds a diverse range of proteins and is associated with a multiplicity of physiological mechanisms.Fig. 2 Reduced lnc-ABCA12-3 expression promotes cell apoptosis. (A-B) Cell apoptosis in lnc-ABCA12-3 silenced or overexpressed Hela cells by flow cytometry (groups of lnc-HIBADH-4 silencing/OE in 200µM 4 h H2O2 treatment) (**P < 0.01). (C) Hochest staining of cell apoptosis in lnc-ABCA12-3 silenced or overexpressed Hela cells. Bule color stained by Hochest 33,342 solution revealed the live cells. Red color stained by PI revealed apoptotic cells. (D-E) Western blot about the level of cleaved-PARP between groups of lnc-HIBADH-4 silencing/overexpression and controls in Hela cells (*P < 0.05).

To gain insights into the biological pathways and interacting proteins associated with lnc-ABCA12-3, we performed RNA pull-down followed by mass spectrometry (MS). RNA pull-down is a well-established technique for studying RNA-protein interactions and its use in lncRNA research can assist us in precisely targeting the proteins on which lncRNAs act. It involves producing a biotin-labeled lnc-ABCA12-3 RNA probe, incubating it with a cytoplasmic protein extract to form RNA-protein complexes, isolating these complexes using streptavidin-coated magnetic beads, and eluting any specifically bound proteins. Following lnc-ABCA12-3 RNA pull-down, silver staining and MS analysis identified 62 unique proteins enriched in the pulldown sample compared to controls, indicating direct or indirect interactions with lnc-ABCA12-3 (supplemental Fig. 1 C-1D).

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of all proteins pulled down by lnc-ABCA12-3 revealed enrichment of several key terms (Fig. 3). GO analysis showed lnc-ABCA12-3 associates with proteins involved in biological processes like protein targeting/transport, as well as cellular components like the cytoplasm and extracellular region (Fig. 3A and B). Molecular function analysis indicated enrichment of proteins with rRNA and cofactor binding abilities (Fig. 3C). KEGG pathway analysis highlighted proteasome, ribosome, and endoplasmic reticulum protein processing as highly enriched pathways (Fig. 3D). These results provide insights into fundamental biological pathways and molecular functions that lnc-ABCA12-3 may regulate through its interacting proteins.Fig. 3 Bioinformatics analysis of lnc-ABCA12-3 pulled down proteins. (A-C) GO analysis of lnc-ABCA12-3 pulled down whole proteins. (D) KEGG analysis of lnc-ABCA12-3 pulled down whole proteins.

4. Lnc-ABCA12-3 binds to UBQLN1, while affects UBQLN1 mRNA and protein expression.

Given the enrichment of proteasome and endoplasmic reticulum protein processing pathways in the GO and KEGG analyses, we focused on one interacting protein in particular, UBQLN1. Ubiquilins (UBQLNs) are a family of cytosolic shuttle proteins that facilitate the degradation of ubiquitinated cargo via proteasome, potentiating autophagy-mediated degradation and contributing to endoplasmic reticulum-associated protein degradation (ERAD)23. UBQLN1 is the predominant ubiquilin isoform wildly expressed among UBQLN families24. It functions as an adaptor between ubiquitinated substrates and the proteasome, enhancing protein degradation25. UBQLN1 also interacts with autophagy machinery by binding LC3 and GABARAP proteins to promote autophagosome formation. Knockdown of UBQLN1 inhibits this process26. Additionally, UBQLN1 binds mTOR and modulates endoplasmic reticulum stress responses27,28. Neurodegenerative disease links further motivated investigating UBQLN1. In vivo mouse studies show UBQLN1 overexpression reduces endoplasmic reticulum stress. Furthermore, mutations in the closely related UBQLN2 gene are associated with familial ALS and frontotemporal dementia in humans29. Given UBQLN1’s established roles in protein quality control via proteasome recruitment, autophagy modulation, and endoplasmic reticulum stress mediation - all pathways implicated in neurodegeneration - we selected it as a prime candidate for elucidating how lnc-ABCA12-3 might regulate cellular proteostasis and influence ALS pathogenesis.

To begin elucidating the molecular interplay between lnc-ABCA12-3 and UBQLN1, we first characterized lnc-ABCA12-3 subcellular localization using RNA fluorescence in situ hybridization (RNA FISH). This technique uses multiple singly labeled oligonucleotide probes against the target RNA to enable visualization via fluorescence microscopy. In HeLa cervical carcinoma cell line, RNA FISH revealed lnc-ABCA12-3 is predominantly localized to the cytoplasm (Fig. 4A), consistent with its potential to interact with the cytosolic protein UBQLN1.Fig. 4 FLnc-ABCA12-3 locates on cytoplasm and binds to UBQLN1, while affects UBQLN1 mRNA and protein expression. (A) Cellular localization of lnc-ABCA12-3 in HeLa cells by RNA FISH (scale bar: 10 μm). (B) Western blot of UBQLN1 expression in Hela cells incubated with or without lnc-ABCA12-3 probe in Hela cells. (C-D) The UBQLN1 mRNA and protein expression change after lnc-ABCA12-3 dysregulation in Hela cells (*P < 0.05, **P < 0.01).

Next, to validate their association, we performed RNA immunoprecipitation assays. Cell lysates were incubated with biotinylated antisense oligonucleotides against either lnc-ABCA12-3 or a nonspecific control. Immunoblotting of pulled down complexes demonstrated lnc-ABCA12-3 binding to UBQLN1 (Fig. 4B). To then assess the impact of modulating lnc-ABCA12-3 levels, we silenced or overexpressed it in cells and measured UBQLN1 expression changes. Both RT-qPCR and Western blot analyses showed knockdown of lnc-ABCA12-3 significantly reduced UBQLN1 mRNA and protein levels. In contrast, lnc-ABCA12-3 overexpression produced only mild upregulation (Fig. 4 C-E). Taken together, these results establish an interaction between lnc-ABCA12-3 and UBQLN1, suggesting lnc-ABCA12-3 may help maintain adequate UBQLN1 expression through a transcriptional and post-transcriptional regulatory mechanism.

5. Lnc-ABCA12-3 reduction affects ubiquitylation and protein processing.

To assess the effects of lnc-ABCA12-3 modulation on protein processing and quality control pathways, we analyzed key markers by Western blotting. Silencing lnc-ABCA12-3 significantly increased phosphorylation of eIF2α (p-eIF2α), an indicator of impaired protein translation initiation (Fig. 5A). However, levels of the endoplasmic reticulum stress proteins GRP78 and Chop were unchanged by lnc-ABCA12-3 knockdown, suggesting no effect on ER stress pathways (supplemental Fig. 2A). Overexpression had no impact on either p-eIF2α or ER stress markers (Fig. 5B, supplemental Fig. 2B). Given UBQLN1’s role in ubiquitin-proteasome system functioning, we also measured total cellular ubiquitination. Knockdown of lnc-ABCA12-3 led to a decrease in ubiquitinated proteins, reflecting impaired protein degradation (Fig. 5C). Conversely, lnc-ABCA12-3 overexpression did not alter ubiquitination.Fig. 5 Lnc-ABCA12-3 reduction affects protein processing and ubiquitination. (A-B) Western blot of phosphor-EIF2a with lnc-ABCA12-3 silencing and overexpression versus controls in Hela cells (*P < 0.05, **P < 0.01). (C) Western blot of whole cell lysate ubiquitination with lnc-ABCA12-3 silencing and overexpression versus controls in Hela cells (***P < 0.001).

Together, these results demonstrate lnc-ABCA12-3 loss specifically disrupts protein processing at the translation level and proteasomal degradation steps of quality control, in line with its interaction with and regulation of UBQLN1 expression. However, endoplasmic reticulum stress branches remain unperturbed. These effects support a model whereby lnc-ABCA12-3 aids neuronal proteostasis maintenance through UBQLN1.

6. Lnc-ABCA12-3 reduction affects autophagic flux, mTOR pathway, and may lead the stress granule formation.

To examine autophagy changes upon lnc-ABCA12-3 perturbation, we employed multiple assays measuring autophagic flux under lnc-ABCA12-3 knockdown. Western blot analyses of lnc-ABCA12-3 silenced LC3-II/I conversion levels comparing to control demonstrated knockdown of lnc-ABCA12-3 impaired autophagosome maturation (Fig. 6A). Moreover, we observed no matter inhibit autophagy by bafilomycin A1 or induced autophagy by oxidative peroxide pharmaceutical H2O2, silencing lnc-ABCA12-3 could lead to suppression of LC3-II/I conversion comparing to control group (Fig. 6B C). Moreover, we provided an mRFP-GFP-LC3 stably expressed U2OS cell model to observe the process of autophagosome and autolysosome after lnc-ABCA12-3 knockdown. It revealed that mRFP + GFP + double-positive spots increased in bafilomycin A1 treated control samples compared to non-treated one, however silencing lnc-ABCA12-3 reduced this accumulation of mRFP + GFP + double-positive spots under bafiomycin A1 treated condition (Fig. 6D). These results indicated suppression of autophagic flux by silencing lnc-ABCA12-3. Unexpectedly, mTOR pathway signals also decreased with lnc-ABCA12-3 loss based on reduced phosphorylation of p70 S6 kinase and S6 ribosomal protein (Fig. 6E). However, autophagy is known to engage both mTOR-dependent and -independent pathways30. It is possible that by interacting with and regulating UBQLN1 levels, lnc-ABCA12-3 modulates autophagy upstream of lysosomal degradation through an mTOR-independent mechanism involving modulation of autophagosome formation. Further studies are warranted to dissect the precise signaling components involved.Fig. 6 Lnc-ABCA12-3 expression affects autophagic flux and mTOR pathway. (A) Western blot of LC3-II/I conversion with lnc-ABCA12-3 silencing versus controls in Hela cells (*P < 0.05). (B-C) Western blot of LC3-II/I conversion in Hela cells between groups of lnc-ABCA12-3 knockdown and controls in the condition of bafilomycin A1 treatment or H2O2 treatment. Bafilomycin A1: 200nM 4 h, H2O2 : 1mM 2 h. (D) mRFP-GFP-LC3 fluorescent staining of U2OS-mRFP-GFP-LC3 stably expressed cells with lnc-ABCA12-3 knockdown. Bafilomycin A1: 200nM 4 h, scale bar: 20 μm. (E) Western blot of phosphor-p70s6k and phosphor-s6k with lnc-ABCA12-3 silencing versus controls in Hela cells (*P < 0.05).

Nonetheless, based on corroborating flux and imaging data, these results collectively demonstrate silencing lnc-ABCA12-3 impair autophagic activity, a critical homeostatic mechanism, through UBQLN1-associated mechanisms. Given the importance of proper protein homeostasis and its link to protein aggregation, we next investigated the impact of lnc-ABCA12-3 knockdown on stress granule formation. Immunofluorescence staining revealed accumulation of cytoplasmic G3BP-positive stress granules in lnc-ABCA12-3 silenced cells, mimicking the induction of stress granules by H2O2 treatment in control cells (Fig. 7). These data indicate that reduced lnc-ABCA12-3 expression influences protein homeostasis by compromising UBQLN1-related pathways and stimulating stress granule assembly.Fig. 7 Reduction of lnc-ABCA12-3 induces stress granules formation. U2OS cells with NC siRNA or lnc-ABCA12-3 siRNA transfection were treated with 1 µM H2O2 for 2 h (to induce G3BP aggregation), fixed, permeabilized and stained with antibodies against G3BP. Scale bar, 50 μm.

Discussion

ALS is a complex neurodegenerative disease with both environmental and genetic risk factors contributing to pathogenesis. However, the specific mechanisms underlying motor neuron damage and disease progression remain elusive. Our study aimed to address this knowledge gap by investigating the potential pathogenic role of the lncRNA lnc-ABCA12-3. Non-coding RNAs, including lncRNAs, have emerged as important regulators of diverse biological processes like tumorigenesis, aging, and neuronal function10,31–33. They may influence disease by modulating target gene expression or interacting with proteins and complexes. Previous microarray analyses and intracellular bioinformatic analysis of peripheral blood monoclonal cells from ALS patients found dysregulated expression of numerous lncRNAs, suggesting lncRNAs may participate in ALS pathogenesis21. However, a few studies had experimentally explored lncRNA functions in ALS prior to this work. This study identified lnc-ABCA12-3 as a potential lncRNA biomarker dysregulated in ALS blood samples, implicating its involvement in disease pathogenesis as a factor influencing development. Our further experiments provided novel insights into potential disease-modifying roles and molecular mechanisms of lnc-ABCA12-3. We confirmed that reduced expression of the lncRNA biomarker lnc-ABCA12-3 correlates with faster disease progression and shorter estimated patient survival times, suggesting its dynamic involvement in ALS pathogenesis and progression over time.

Lnc-ABCA12-3 is not a novel lncRNA in the context of disease-associated studies. Previous research has investigated the role and mechanism of lnc-ABCA12-3 in esophageal squamous cell carcinoma pathogenesis. In these studies, lnc-ABCA12-3 expression was evaluated in esophageal squamous cell carcinoma samples and found to regulate cell migration, invasion, and proliferation abilities by acting as a competing endogenous RNA for miR-200b-3p to modulate FN1 expression22. More recent work demonstrated lnc-ABCA12-3 can promote proliferation and glycolysis in esophageal cancer cells while repressing apoptosis, potentially via modulating the TLR4/NF-κB signaling pathway under exosomal mediation34. Our current study aimed to characterize lnc-ABCA12-3 in the context of ALS for the first time.

We utilized vitro experimental techniques to validate and explore the functional role and downstream pathways associated with lnc-ABCA12-3. Cell-based assays characterized the effects of modulating lnc-ABCA12-3 levels on cellular phenotypes relevant to ALS pathogenesis like apoptosis and protein homeostasis. Mechanistically, we sought to explore the direct protein interactome of lnc-ABCA12-3 using established RNA-protein interaction techniques. RNA-protein pull-down assays and RNA immunoprecipitation allow for more effective identification of lncRNA-associated proteins and biological processes. In RNA-protein pull-down assays, biotin-labeled in vitro transcribed lnc-ABCA12-3 RNA was bound to streptavidin beads and incubated with cell lysates to capture interacting proteins. Alternatively, RIP utilizes an antibody against a protein of interest to co-immunoprecipitate any endogenous RNAs in complex35. Gene ontology and pathway analysis of these interacting proteins provided several key insights. These results provide clues to potential roles of lnc-ABCA12-3 in protein transport, localization, as well as involvement in fundamental processes like transcription and translation. Further investigation of top candidate interacting proteins may help elucidate the specific mechanisms through which lnc-ABCA12-3 functions. Lnc-ABCA12-3 appeared to maintain important protein homeostasis pathways, such as ubiquitination and autophagy, through regulating the expression and activity of its interactor UBQLN1.

Dysregulated protein homeostasis, also known as proteostasis, is a hallmark of numerous neurodegenerative diseases. The proteostasis network comprises multiple interconnected systems that work cooperatively to ensure proper folding, trafficking, and clearance of proteins in neurons36. This network includes molecular chaperones and co-chaperones that facilitate protein folding, the ubiquitin-proteasome system that degrades ubiquitinated proteins, autophagy pathways for the lysosomal degradation of protein aggregates and damaged organelles, as well as components involved in stress granule dynamics37. Under stress conditions, misfolded proteins accumulate when these proteostasis mechanisms are impaired. Defects in maintaining proteostasis have been strongly implicated in the pathogenesis of various neurodegenerative diseases by facilitating aggregation of disease-associated proteins and compromising neuronal integrity over time38. Extensive research has identified roles for numerous noncoding RNAs, including microRNAs, rRNAs and lncRNAs, in modulating protein homeostasis at post-transcriptional and translational levels39–43. Disruptions in homeostasis have been linked to neurodegenerative conditions like ALS, Huntington’s disease and Alzheimer’s disease through diverse mechanisms. Ubiquilins (UBQLNs) are multifunctional shuttle proteins that facilitate degradation of misfolded proteins via the ubiquitin-proteasome system, autophagy and ER-associated degradation pathways44–46. Moreover, disruption of ubiquitin signaling and accumulation of misfolded/aggregated proteins are hallmarks of these diseases. Meanwhile, UBQLN protein expression levels contribute to roles of UBQLNs in these processing and in the progression of several diseases, however regulatory mechanisms for UBQLN expression levels remain unclear47. In this study, we confirmed binding of lnc-ABCA12-3 to UBQLN1 using established RNA-protein interaction techniques. Dysregulated lnc-ABCA12-3 was found to influence UBQLN1-associated pathways like ubiquitination, autophagy, and mTOR, likely through regulating protein levels. We also found the induced stress granule formation in lnc-ABCA12-3 down expressed cells. We demonstrated lnc-ABCA12-3 modulates UBQLN1 expression at both the mRNA and protein levels. This finding highlights the dual regulatory role of lnc-ABCA12-3 on UBQLN1 expression, impacting both transcriptional and post-transcriptional levels. Our data shows that knockdown of lnc-ABCA12-3 reduces UBQLN1 mRNA levels, suggesting a role in transcriptional regulation. This observation is supported by our RT-qPCR results, where a significant reduction in UBQLN1 mRNA was observed upon lnc-ABCA12-3 knockdown. Additionally, we observed changes in UBQLN1 protein levels in response to lnc-ABCA12-3 knockdown and overexpression, indicating a post-transcriptional regulatory mechanism. The RNA pull-down assays and RNA immunoprecipitation further support that lnc-ABCA12-3 interacts with UBQLN1, suggesting a role in modulating UBQLN1 protein stability or translation. However, the precise molecular mechanism by which lnc-ABCA12-3 regulates UBQLN1 mRNA remains to be elucidated. Still, these findings implicate lnc-ABCA12-3-mediated control of UBQLN1 dysfunction in ALS pathogenesis.

While this study provides novel insights into lncRNA involvement in ALS pathogenesis, several limitations must be acknowledged. The present study only analyzed patients within one year of symptom onset, representing a limitation. In future studies, longitudinal collection and analysis of long-term patient follow-up data would allow better observation of lnc-ABCA12-3 dynamic changes throughout the ALS disease course. This would facilitate more comprehensive and in-depth understanding of its role in ALS pathogenesis. It is also important to expand sample sizes to include cases with different survival periods. Additionally, our studying one lncRNA-protein pair provides valuable preliminary mechanistic insights but does not capture the complexity of molecular networks in the ALS context. Most neurodegenerative diseases involve dysregulation of many noncoding and coding genes interacting in pathways, so one lncRNA is unlikely to be solely responsible or representative11,21. The specific regulatory mechanism elucidated may not apply to other lncRNA-target relationships in the same or related diseases. Broader lncRNA and protein profiling approaches like CLIP-seq, RIP-seq could systematically map the interactome wired by lnc-ABCA12-3 and related transcripts. This would provide a more holistic view of molecular circuitry involvement and help pinpoint key hub or driver molecules versus minor players. Our studies have also focused on peripheral blood cells and general cell lines, neglecting key neuron and glia populations relevant to ALS pathogenesis. Single-cell profiling of central and peripheral tissues could identify cell-type specific lnc-ABCA12-3 functions and understand local vs. systemic pathway involvement. Comparisons between CNS motor regions and other affected areas would also strengthen tissue-level conclusions.

Conclusion

In this study, we employed a comprehensive “clinic to bench” research approach to unravel the molecular mechanisms implicated in the dysregulation of lncRNA in Amyotrophic lateral sclerosis (ALS). The integration of clinical sample analyses and molecular examination has unveiled unique mechanistic insights into how the dysregulation of the target lncRNA, lnc-ABCA12-3, might be intertwined with ALS disease pathways. Notably, we found that lnc-ABCA12-3 significantly influences the expression of the ubiquitin shuttle protein UBQLN1, a key player in maintaining protein homeostasis, which is crucial in the pathogenesis of ALS. This multi-faceted investigation has broadened our understanding of lncRNA’s role in ALS, and positions lnc-ABCA12-3, along with its interaction with UBQLN1, as potential biomarkers and therapeutic targets deserving of further scrutiny. Our study lays the groundwork for ongoing clinicopathological correlation studies concerning lncRNAs and their protein interactors, like UBQLN1, in the context of neurodegenerative diseases.

Methods

Clinical sample collection

We obtained peripheral blood mononuclear cell (PBMC) samples from 60 amyotrophic lateral sclerosis (ALS) patients, 40 age-sex matched healthy controls (HCs), and 40 Parkinson’s disease (PD) patients who were recruited from West China Hospital. The clinical characteristics of the patients have been detailed in our previous study, for which ethical approval was granted by the Institutional Review Board of West China Hospital21. All participants, including patients and HCs, provided written informed consent. PBMCs were extracted from whole blood using standard methods as described previously. In brief, blood samples were collected into vacutainer tubes containing EDTA as an anticoagulant. PBMCs were then isolated by density gradient centrifugation and washed twice with phosphate-buffered saline. Total RNA was extracted from the isolated PBMCs using standard protocols to enable further molecular analyses.

Statistical analyses

All experiments were independently repeated at least three times. Data are presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed using SPSS (version 19.0) and GraphPad Prism (version 9.0) software. Differences between multiple groups were evaluated using one-way analysis of variance (ANOVA). The unpaired two-tailed Student’s t-test was used to analyze differences between two groups. A P-value < 0.05 was considered statistically significant. P-values < 0.05, < 0.01, and < 0.001 are denoted as *, **, and ***, respectively.

Calculation of disease progression rate and survival status

The Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS) is a 10-item functional inventory which was devised for use in therapeutic trials in ALS. Each item is rated on a 0–4 scale by the patient and/or caregiver, yielding a maximum score of 40 points. Pearson correlation analysis assessed the relationship between lncRNA expression levels and clinical characteristics. Survival analysis was conducted using the Kaplan-Meier method, and survival differences were determined by the log-rank test. Disease progression calculation as below:

Disease progression rate = 48 * (48 - ALSFRS at blood collection);

6 months disease progression rate = (score at visit - score after 6 month) /6;

12 months disease progression rate = (score at visit - score after 12 month)/12.

Cell culture

The human cervical cancer cell lines HeLa and the human bone osteosarcoma epithelial cells were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). HeLa cells and U2OS cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin solution. All cell lines were incubated at 37℃ in the presence of 5% CO2.

Lentiviral vectors and cell infection

U2OS cells were seeded in 6-well plates at a density of 2 × 10^5^ cells/well and cultured overnight. Cells were then 20 µl mRFP-GFP-LC3 lentivirus (Genechem, Shanghai, China) and 40 µl infectious reagent. After 24 h, the infection medium was replaced with fresh medium. Infection efficiency was assessed at 48 and 72 h post-infection by fluorescence microscopy. Stably transduced cells were selected by culturing in medium containing 2.0 µg/mL puromycin for 48 h, followed by 0.5 µg/mL puromycin for 2 weeks to eliminate untransduced cells. Surviving cells exhibiting red and green fluorescence were maintained for further experiments after verification of LC3 expression by Western blotting. Cells were frozen down in liquid nitrogen and stored in liquid nitrogen tanks for future use.

Reverse transcription and quantitative real-time PCR (RT-qPCR)

Total RNA was extracted from cells/tissue using TRIzol reagent (Takara, Kyoto, Japan) according to the manufacturer’s instructions. RNA concentration and purity were determined by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Subsequently, cDNA was synthesized from 1 µg of total RNA using the Prime-Script RT Reagent Kit (Takara). Real-time quantitative PCR (qPCR) was then performed on an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific) using TB Green Premix Ex Taq II (Takara). Thermal cycling conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. The 2 − ΔΔCt method was used to analyze relative gene expression levels, which were normalized to the endogenous control GAPDH. Primer sequences are listed in Supplementary Table 1.

RNA pull down and HPLC-MS/MS high performance liquid chromatography with Mass Spectrometry

RNA pull down and HPLC-MS/MS experiments were performed to identify proteins that interact with lnc-ABCA12-3 by GeneCreate (Wuhan, China) according to the manufacturer’s instructions. HeLa cells were transfected with the lnc-ABCA12-3 overexpression plasmid or empty vector control. Cell lysates were incubated with in vitro transcribed biotin labeling lnc-ABCA12-3 or NC probes. Designed and synthesized lncRNA probe and control probe were listed in Supplementary Table 2. Magnetic beads were used to capture biotinylated RNA-protein complexes. The beads were washed and bound proteins were subjected to SDS-PAGE followed by silver staining. The above complex was incubated with cell lysate to pull down the enriched lncRNA, and at the same time enrich to the protein that binds to lncRNA. The above proteins were subjected to SDS-PAGE gel electrophoresis and silver staining. The whole protein solution pulled down from the experimental group and the control group were separately identified by mass spectrometry to find out the possible differential proteins.

RNA fluorescence in situ hybridization (RNA FISH)

Hela cells were seeded into 12-well plates containing coverslips and cultured for 24 h. Following fixation with 4% paraformaldehyde for 20 min at room temperature, cells were permeabilized with 0.5% Triton X-100 in PBS. Fluorescence in situ hybridization (FISH) was performed using a 2 µM Cy3-labeled lnc-ABCA12-3 probe (stare-FISH Probe Mix, GenePharma, Shanghai, China) according to the manufacturer’s instructions. The probe sequences were listed in Supplementary Table 2. Cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific). Coverslips were mounted onto glass slides using mounting medium. Images were acquired on a fluorescence microscope (Nikon, Tokyo, Japan) at 60× magnification. FISH signal localization was analyzed using ImageJ software.

Cell transfection

Small interfering RNAs (siRNAs) targeting lnc-ABCA12-3 (si-lnc-ABCA12-3) and a negative control siRNA (si-NC) were synthesized by GenePharma (Shanghai, China). The siRNA sequences are listed in Supplementary Table 2. A recombinant plasmid expressing lnc-ABCA12-3 (lnc-ABCA12-3 overexpression vector) and an empty vector control were constructed by Genechem (Shanghai, China). Cells were seeded in 12-well plates at a density of 1 × 105 cells/well and cultured for 24 h. Then, siRNAs or plasmids were transfected into cells using GP-mate transfection reagent (GenePharma, Shanghai, China) according to the manufacturer’s protocol. After 48–72 h of transfection, cells were harvested and subjected to downstream analysis, including quantitative real-time PCR and cell functional assays.

Flow cytometric analyses

Apoptosis was assessed using Annexin V-FITC/PI staining. Briefly, HeLa cells were seeded in 6-well plates and treated with hydrogen peroxide (H2O2; 200 µM) for 2 h. Untreated cells were used as control. After treatment, cells were harvested with 0.25% trypsin (without EDTA) and washed with cold PBS. Annexin V-FITC/PI staining was performed using an Apoptosis Detection Kit (YEASEN) according to the manufacturer’s protocol. Stained cells were analyzed by flow cytometry using a FACSAria III instrument (BD Biosciences, USA). A minimum of 20,000 cells per sample was acquired. The percentages of apoptotic cells (Annexin V+/PI- for early apoptosis; Annexin V+/PI + for late apoptosis) were quantified using FlowJo software.

Hochest staining

Culture medium was removed and coverslips were washed twice with 1X PBS. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Coverslips were washed twice with PBS and stained with 5 µg/mL Hoechst 33,342 solution for 20 min protected from light. Following Hoechst staining, coverslips were incubated with propidium iodide (PI) solution at 1 µg/mL for 20 min protected from light. Finally, the cells were visualized using a fluorescence microscope (Nikon, Tokyo, Japan) at 10× magnification.

Western blot analysis

Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein lysates were separated on 8–15% SDS-PAGE gels and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk for 1 h at room temperature followed by incubation with primary antibodies overnight at 4 °C. Primary antibodies and dilutions are listed in Supplementary Table 3. Membranes were washed and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using substrate (Thermo Fisher Scientific) and imaged using a ChemiDoc imaging system (Bio-Rad). Densitometry analysis was performed using ImageJ software. Where indicated, cells were treated with 200nM bafilomycin A1 (MedChemExpress) for 4 h to inhibit autophagy prior to protein extraction. Equal loading was assessed using tubulin or beta-actin.

Immunofluorescence staining

U2OS cells transfected with siNC or si-lnc-ABCA12-3 were seeded on coverslips in 12-well plates. After 48 h, cells were treated with 200 µM H2O2 for 2 h. Cells were then fixed with 4% paraformaldehyde for 20 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min. Following blocking with 5% goat serum for 30 min, cells were incubated overnight at 4 °C with anti-G3BP1 primary antibody (1:200, Proteintech) dilution in 3% BSA. Coverslips were washed 3 times with PBS and incubated with Alexa Fluor 594-conjugated secondary antibody (Thermo Fisher Scientific) in the dark for 1 h at room temperature. DAPI was used to counterstain nuclei. Finally, the cells were visualized using a fluorescence microscope (Nikon, Tokyo, Japan) at 40× magnification.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2

Supplementary Material 3

Supplementary Material 4

Supplementary Material 5

Supplementary Material 6

Supplementary Material 7

Supplementary Material 8

Supplementary Material 9

Supplementary Material 10

Supplementary Material 11

Supplementary Material 12

Supplementary Material 13

Abbreviations

ALS Amyotrophic lateral sclerosis

NcRNAs non-coding RNAs

LncRNAs long non-coding RNAs

RT-qPCR reverse transcription-quantitative real-time PCR

GO Gene Oncology

KEGG Kyoto Encyclopedia of Genes and Genomes

MS mass spectrum

Acknowledgements

The authors thank all subjects for their participation in the study.

Author contributions

YJY searched and selected the studies, analyzed the data, drafted and revised the article. DJP, JXH, and YYC prepared the samples and gave suggestions on study design. CYL and HFS designed the study and gave suggestions on revising the article. All authors read and approved the final manuscript.

Funding

The present study was supported by the funding of The National Natural Science Foundation of China (Grant No. 81871000), The Sichuan Science and Technology Program (Grant No.2022NSFSC0750) and The China Postdoctoral Science Foundation (Grant No. 2021M692296). The National Natural Science Foundation of China (Grant No. 81871000), Huifang Shang. The Sichuan Science and Technology Program (Grant No.2022NSFSC0750), Dejiang Pang. The China Postdoctoral Science Foundation (Grant No. 2021M692296), Yujiao Yu.

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

West China Hospital approved the study, and all participants gave signed informed consent. This study was conducted in accordance with the Declaration of Helsinki and has been passed ethical approval from the Institutional Review Board of West China Hospital.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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