![Analysis of the hsa-miRNA-30b-5p (miRNA-30b) interaction with the Homo sapien NF-L 3'-UTR; (A) representation of the nucleotide complementarity between the 22 nucleotide (nt) hsa-miR-30b-5p (highlighted in green; encoded at the miRNA-30 gene cluster on human chromosome (chr) 8q24.22; https://www.genecards.org/cgi-bin/carddisp.pl?gene=MIR30B) and nt position 266–287 of the NF-L mRNA 3'-UTR non-coding region (highlighted in yellow; encoded at human chr 8p21.1; https://www.genecards.org/cgi-bin/carddisp.pl?gene=NEFL); the microRNA target prediction database (miRDB; http://mirdb.org/cgi-bin/targetdetail.cgi?targetID=2099169; last accessed April 24, 2022) for miRNA-30b and NF-L (NEFL; NCBI Gene ID 4747; GenBank Accession NM_006158) indicates a very high miRNA-mRNA target score of 84 and a strong 10 nt ‘seed' sequence location at 278-287 nt of the NF-L 3'-UTR [see also “(B)” below]; (B) the NF-L 3'-UTR gene sequence; the inverted red arrow indicates the start of the NF-L 3'-UTR non-coding sequence; last upward pointing red arrow is the end of the NF-L 3'-UTR; note that additional adenosine groups are present in the mature NF-L mRNA (and 3' end of the NF-L 3'-UTR); (C) the NF-L-mRNA-3′-UTR expression vector luciferase reporter assay (pLight Switch-3′UTR; Cat#S810535; Switchgear Genomics, Palo Alto CA); in this vector, the entire 1,985 nucleotide NF-L 3′-UTR was ligated into the unique Nhe1-Xho1site; not drawn to scale; (D) human neuronal-glial (HNG) cells, 2 weeks in primary culture; neurons (red stain; λmax = 690 nm), DAPI (blue nuclear stain; λmax = 470 nm) and glial fibrillary associated protein (GFAP; glial-specific green stain; λmax = 520 nm); the HNG cell culture is about 60% confluent and at 2 weeks of culture contains about 70% neurons and 30% astroglia (7, 14, 18, 39, 40, 49); human neurons do not culture well in the absence of glia; neurons also show both extensive cytoarchitecture and display electrical activity (unpublished; Lonza Research and Development, Walkersville MD, USA); 40X magnification; HNG cells transfected with the NF-L-mRNA-3′-UTR expression vector luciferase reporter were treated exogenously with LPS (20 ng/ml cell culture medium, 48 hr), a stabilized miRNA-30b, a scrambled control miRNA-30b (miRNA-30b-sc) or control miRNA-183; see (14, 18, 39, 49) and text for further details on all reagents and methods used in these experiments; (E) compared to control, HNG cells transfected with a scrambled (sc) control pLightSwitch-3'-UTR vector, the NF-L-mRNA-3′-UTR vector exhibited decreased luciferase signal to a mean of 0.18-fold of controls in the presence of exogenous LPS and 0.11 in the presence of miRNA-30b; this same vector exhibited no change in relative luciferase yield in the presence of a control miRNA-30b-sc or miRNA-183; for each experiment (using different batches of HNG cells) a control luciferase signal was generated that included separate controls with each analysis; in addition a control vector β-actin-3′-UTR showed no significant effects on the relative luciferase signal yield after treatment with either miRNA-183 or miRNA-30b (data not shown); a dashed horizontal line set to 1 is included for ease of comparison; N = 5; *p < 0.01 (ANOVA); values represent mean +/- 1 standard deviation (S.D.); Microsoft Excel Analysis ToolPak, Excel for Microsoft 365; https://support.microsoft.com/en-us/office/use-the-analysis-toolpak-to-perform-complex-data-analysis-6c67ccf0-f4a9-487c-8dec-bdb5a2cefab6. The results suggest a physiologically relevant miRNA-30b-NF-L-mRNA-3′-UTR interaction and a miRNA-30b-mediated downregulation of NF-L expression in HNG cells. This pathogenic interaction may be related to the downregulation of other immune, inflammatory, and synaptic system genes by upregulated miRNAs in the CNS resulting in a deficit in cytoskeletal and synaptic organization and trans-synaptic signaling (7, 21, 26, 27, 31, 32, 36, 38–40, 50).](https://www.frontiersin.org/_rtmag/_next/image?url=https%3A%2F%2Fwww.frontiersin.org%2Ffiles%2FArticles%2F900048%2Ffneur-13-900048-HTML%2Fimage_m%2Ffneur-13-900048-g001.jpg&w=3840&q=75)
Microbiome-derived Gram-negative bacterial lipopolysaccharide (LPS) has been shown by multiple laboratories to reside within Alzheimer's disease (AD)-affected neocortical and hippocampal neurons. LPS and other pro-inflammatory stressors strongly induce a defined set of NF-kB (p50/p65)-sensitive human microRNAs, including a brain-enriched Homo sapien microRNA-30b-5p (hsa-miRNA-30b-5p; miRNA-30b). Here we provide evidence that this neuropathology-associated miRNA, known to be upregulated in AD brain and LPS-stressed human neuronal-glial (HNG) cells in primary culture targets the neurofilament light (NF-L) chain mRNA 3'-untranslated region (3'-UTR), which is conducive to the post-transcriptional downregulation of NF-L expression observed within both AD and LPS-treated HNG cells. A deficiency of NF-L is associated with consequent atrophy of the neuronal cytoskeleton and the disruption of synaptic organization. Interestingly, miRNA-30b has previously been shown to be highly expressed in amyloid-beta (Aβ) peptide-treated animal and cell models, and Aβ peptides promote LPS entry into neurons. Increased miRNA-30b expression induces neuronal injury, neuron loss, neuronal inflammation, impairment of synaptic transmission, and synaptic failure in neurodegenerative disease and transgenic murine models. This gut microbiota-derived LPS-NF-kB-miRNA-30b-NF-L pathological signaling network: (i) underscores a positive pathological link between the LPS of gastrointestinal (GI)-tract microbes and the inflammatory neuropathology, disordered cytoskeleton, and disrupted synaptic signaling of the AD brain and stressed brain cells; and (ii) is the first example of a microbiome-derived neurotoxic glycolipid having significant detrimental miRNA-30b-mediated actions on the expression of NF-L, an abundant neuron-specific filament protein known to be important in the maintenance of neuronal cell shape, axonal caliber, and synaptic homeostasis.
