ALS is a complex disease believed to be caused by environmental and genetic factors, but the specific mechanism remains elusive[33]. Non-coding RNA, a large class of RNA, plays an important role in tumorigenesis, aging, and may serve as a potential drug target[34], [35]. Previous study of microarray assay for peripheral blood mononuclear cells of sALS and HC showed that the expression of RNAs that changed between sALS and HC were mostly non-coding RNA, especially lncRNAs[36]. These results suggest that non-coding RNA may play an important role in the pathogenesis of ALS. However, few studies have explored the function of lncRNAs in ALS experimentally[37, 38]. Our study revealed that lnc-HIBADH-4 is significantly downregulated in ALS patients and correlates with disease severity and overall survival, which further confirm the role of lncRNAs in ALS. Lnc-HIBADH-4 regulates lysosomal function by competitively sponging miR-326, which increases CTSD expression and participates in proliferation and apoptosis. (Fig. 6).
We identified and validated lnc-HIBADH-4 as one of the most significantly downregulated lncRNAs in ALS patients. Several miRNAs[14, 18], including miR-181, have been found to be differentially expression in peripheral blood cells and may serve as novel biomarkers when combined with NfL[13]. Although several studies have investigated the mechanisms of lncRNAs in the pathogenesis of ALS, their potential as diagnostic and prognostic biomarkers remain unknown[38–40]. Our study demonstrated that decreased lnc-HIBADH-4 expression was negatively correlated with disease severity and survival in ALS patients. Lnc-HIBADH-4 is an intronic lncRNA of JAZF1, located in chromosome 7, and its biological function in ALS has not been explored previously. Therefore, it is essential to investigate the role of lnc-HIBADH-4 in ALS pathogenesis.
We found that silencing lnc-HIBADH-4 resulted in increased cell apoptosis and decreased cell proliferation, particularly under conditions of oxidative stress and apoptosis-inducing stimuli. These findings suggest that decreased expression of lnc-HIBADH-4 is detrimental to cells and may contribute to the pathogenesis of ALS. We further investigated the mechanisms underlying the effects of lnc-HIBADH-4 on cell function and observed that downregulation of lnc-HIBADH-4 inhibited the autophagy-lysosomal pathway, particularly by impairing the formation of autophagolysosomal and inducing lysosomal dysfunction. Lysosomes play a crucial role in the autophagy pathway by degrading cytosolic proteins and organelles, and CTSD is a key protease involved in lysosomal function[4]. CTSD is widely expressed in neurons and is essential for the clearance of protein aggregates and neuroprotection[26, 41]. Decreased expression of CTSD in neurons results in the accumulation of proteins and impairs lysosomal activity, particularly in neurodegenerative diseases such as ALS[26, 42, 43]. Studies have shown that CTSD levels are reduced in spinal motor neurons of TDP-43 knockout mice following lysosomal dysfunction, while in sALS and SOD1G93A mice, CTSD mRNA expression increases gradually with disease progression[44], [45]. Although the spinal cord of SOD1G93A mice showed increased expressions of CTSD, but CTSD were almost cleaved and without normal function in lysosome[46]. These findings demonstrate the critical role of CTSD in lysosomal function in ALS and suggest that the expression of CTSD mRNA may increase in response to lysosomal dysfunction in ALS patients and models. Both decreased expression and dysfunction of CTSD can impact the pathophysiological process of ALS, underscoring the importance of maintaining normal levels and function of CTSD in neurons. Therefore, developing therapies that target CTSD and other components of the lysosomal pathway holds promise for treating ALS and other neurodegenerative diseases.
Our research found that knockdown lnc-HIBADH-4 resulted in decreased expression of CTSD and lysosomal dysfunction. However, the mechanism underlying the link between lnc-HIBADH-4 and CTSD expression required further investigation. Considered that lnc-HIBADH-4 is located in the cytoplasm primarily, where miRNAs frequently interact with lncRNAs and mRNAs, therefore, we examined the potential role of miRNAs in mediating the relationship between lnc-HIBADH-4 and CTSD. We identified miR-326 as a potential bridge between lnc-HIBADH-4 and CTSD. We discovered a negative correlation between miR-326 and lnc-HIBADH-4 expression levels, and found that miR-326 was significantly upregulated in ALS patients, consistent with prior research conducted an Italian group[47]. MiR-326 has been suggested as a potential biomarker for neurodegenerative diseases and has been shown to inhibit cell growth and promote cell death in various disease models[48], [49–51]. Studies have reported different roles for miR-326 in autophagy, with different target genes being identified for miR-326[52, 53]. To our knowledge, this is the first study to identify a relationship between miR-326 and lysosomal function. Taken together, our findings suggest that lnc-HIBADH-4/miR-326 represents a potential therapeutic target for regulating CTSD expression in ALS. Future studies aimed at further elucidating the roles of lnc-HIBADH-4 and miR-326 in the pathogenesis of ALS may lead to the development of novel therapeutic approaches for the treatment of this devastating disease.
Our study has several limitations that should be addressed in the future research. Firstly, while we focused on the expression of lnc-HIBADH-4 in ALS patients, there are numerous other lncRNAs that are differentially expressed in ALS. It remains unclear whether other lncRNAs are involved in the pathogenesis of the disease[10]. Secondly, our investigation of lnc-HIBADH-4 was conducted in vitro, using HeLa cells as a model system. However, due to the low conservation of lnc-HIBADH-4 (no expression in mouse and other non-primates), it is necessary to study its role in vivo (primates) and in motor neuron cells derived from induced pluripotent stem cells (iPSCs) of ALS patients. Moreover, future studies could investigate the upstream regulatory factors of lnc-HIBADH-4 to identify potential therapeutic targets for maintaining normal levels of lnc-HIBADH-4 expression in cells, which may help prevent the occurrence and progression of ALS.