Downregulation of Neurofilament Light Chain Expression in Human Neuronal-Glial Cell Co-Cultures by a Microbiome-Derived Lipopolysaccharide-Induced miRNA-30b-5p

Abstract

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.

Lipopolysaccharides (LPSs) as Potent Neurotoxic Glycolipids in Alzheimer’s Disease (AD)

“…These pathogenic neurotoxins were found to significantly disrupt normal gene expression patterns in the CNS and include multiple species of Gram-negative bacteria-derived neurotoxic glycolipids long known to be extremely potent inducers of pro-inflammatory and altered immunological signaling in infection, sepsis, cancer and neurological diseases, including AD [ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ]. Recent research evidence indicates that LPS generation, abundance and secretion is stimulated by a number of factors, including: (i) the proliferation of enterotoxigenic LPS-secreting microbes of the GI-tract microbiome and, more specifically, anaerobic Gram-negative bacterial species; (ii) metallotoxic environmentally abundant cellular stressors [ 12 , 29 , 30 ]; and (iii) other lifestyle-related inducers, including those commonly found in ‘unhealthy’ Western diets that include high fat and cholesterol (HF-C) consumption and insufficient dietary fiber intake, and other factors [ 28 , 31 , 32 , 33 , 34 , 35 , 36 ]. It should be kept in mind that neurotoxic biopolymers originating from the human GI-tract microbiome are solely derived from multiple Gram-negative bacterial species; however, under physiological conditions, very complex combinations of LPSs and other neurotoxins are derived from the entire repertoire of GI-tract resident microorganisms [ 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ].…”

“…The genus Bacteroidetes , and in particular Gram-negative anaerobe, non-spore -forming bacillus Bacteroides fragilis , which is especially abundant in deeper regions of the GI-tract, is among the most studied and genetically understood of all human GI-tract resident microbes [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 31 , 32 , 33 , 37 ]. B. fragilis exhibits a significant amount of intra-species genomic diversity and associated range and variety of potential biochemical functions, and as a prominent Genus species , it has significant potential to secrete: (i) both a ‘generic’ form of LPS and a unique, exceptionally potent, pro-inflammatory LPS subtype, BF-LPS [ 30 , 38 ]; (ii) a zinc-metalloproteinase known as B. fragilis toxin (BFT) or fragilysin; (iii) truncated LPS molecules known as lipooligosaccharides ( LOSs ) [ 8 , 19 , 20 ]; and (iv) bacterial-derived miRNA-like small non-coding RNAs (sncRNAs) [ 18 , 19 , 20 , 21 ].…”

“…B. fragilis exhibits a significant amount of intra-species genomic diversity and associated range and variety of potential biochemical functions, and as a prominent Genus species , it has significant potential to secrete: (i) both a ‘generic’ form of LPS and a unique, exceptionally potent, pro-inflammatory LPS subtype, BF-LPS [ 30 , 38 ]; (ii) a zinc-metalloproteinase known as B. fragilis toxin (BFT) or fragilysin; (iii) truncated LPS molecules known as lipooligosaccharides ( LOSs ) [ 8 , 19 , 20 ]; and (iv) bacterial-derived miRNA-like small non-coding RNAs (sncRNAs) [ 18 , 19 , 20 , 21 ]. Bacterial-derived sncRNAs are important microbial regulators that often act to transmit environmental signals when cells encounter suboptimal or stressful growth conditions and whose functions remain incompletely understood [ 29 , 30 , 37 , 38 ]. Importantly, LPSs, BF-LPS and/or BFT ( fragilysin ) have significant capabilities to disrupt paracellular and transcellular barriers via the cleavage of intercellular adhesion proteins, resulting in ‘leaky’ and easily breached biophysical barriers [ 13 , 14 , 15 , 16 , 17 , 18 ].…”

“…GI-tract microbiome dysbiosis or infection by multiple species of toxic bacteria or their secretory products into the brain and CNS was proposed to contribute to the initiation or propagation of the pathogenesis of AD by triggering or accelerating neuro-inflammatory and neurodegenerative responses [ 38 , 40 , 41 , 42 , 43 ]. Less than 6 years ago, several independent reports emerged that detected the presence of GI-tract microbiome-derived LPSs within human brain cells of the CNS both during advanced aging and in the AD brain [ 27 , 28 , 29 , 31 , 32 , 33 , 37 , 43 , 44 , 45 , 46 , 47 , 48 , 49 ]. Interestingly, (i) each Gram-negative bacillus, indeed, each GI-tract resident microbe, has the potential to secrete a slightly different LPS and/or neurotoxin assortment with slightly different lipid and oligosaccharide domain structures, abundances, activities, molecular properties and toxicities [ 17 , 18 , 19 , 20 , 27 ]; (ii) LPSs have a remarkably high affinity for human neocortical neuronal plasma membranes, and this attraction is significantly enhanced in the presence of the amyloid-beta 42 (Aβ42) peptides that accumulate in the AD-affected brain [ 15 , 16 , 17 , 18 , 19 , 20 , 31 , 45 ] ( Figure 1 ); (iii) Gram-negative bacterial molecules associate with AD neuropathology, including amyloid plaques, neurons and oligodendrocytes, in the AD brain [ 41 , 42 , 43 , 44 , 45 , 46 ]; (iv) microbiome-derived E. coli LPSs and BF-LPS are associated with the hippocampal CA1 and neocortical regions in AD brain [ 41 ,…”

“…The most recent evidence of a neuropathological pathway involving GI-tract-microbiome-derived LPSs and/or BF-LPS, beginning with the LS-mediated up-regulation of pro-inflammatory transcription factor NF-kB (p50/p65) and miRNA-30b signaling, and ending with the specific down-regulation of the neuron-specific cytoskeletal NF-L element is noteworthy [ 29 , 39 ]. This pathway: (i) for the first time, underscores a positive pathological link between the LPSs of GI-tract microbes and the disordered cytoskeleton and disrupted synaptic signaling of the AD brain and stressed human neuronal–glial (HNG) cells in primary culture; (ii) in part characterizes the up-regulation of NF-kB (p50/p65), a brain-abundant transcription factor known to support inflammatory neuropathology; (iii) is representative of a local and progressive pathological signaling system along the gut–brain axis with potential to operate over the life-span of Homo sapiens ; and (iv) it is the first example of a microbiome-derived neurotoxic glycolipid having significant detrimental microRNA-mediated actions on the expression of NF-L, an abundant neuron-specific filamentous protein known to be important for the maintenance of neuronal cell structure and shape, and synaptic homeostasis [ 27 , 39 , 53 ].…”