Fig 1: Studies of LPS-neuronal nuclear binding in HNG cells in primary culture. (A) human neuronal-glial (HNG) cells in primary co-culture at 2 weeks; neurons (red stain; λmax = 690 nm), DAPI (blue nuclear stain; λmax = 470 nm) and GFAP (glial-specific green stain; λmax = 520 nm); human neurons do not culture well in the absence of glia; neurons also show both extensive arborization and display electrical activity (unpublished; Lonza); scale bar = 20 μm; (B) details of association of LPS (red stain; λmax = 690 nm) and nuclear DAPI (blue stain; λmax = 470 nm); note affinity of red-stained LPS with blue-stained nuclei after only 48 h of co-incubation (arrows); see also Supplementary File 1 (Details of accumulation of LPS in HNG cells in primary culture); scale bar for all photos (lower right) = 10 μm; (C) Neurofilament heavy, medium and light (NF-H, NF-M, and NF-L) chain abundance in control and LPS-treated HNG cells – cluster analysis of gene expression (mRNA levels); in two controls (HNG-1 and HNG-2) and in two LPS-treated samples (LPS-1, LPS-2), LPS-treated HNG cells exhibit a marked reduction in NF-L expression, a reduction that is not as apparent in NF-H or NF-M expression; NF-H, NF-M, and NF-L expression was quantified against the levels of β-actin and GAPDH in the same sample; (D) samples are quantified in bar graph format showing the mean and one standard deviation of all three neurofilament protein levels; there was no statistically significant change in NF-H, NF-M, β-actin, or GAPDH between control and LPS-treated HNG cells, however NF-L levels were reduced to about 0.22-fold of controls in LPS-treated HNG cells; interestingly the NF-H, NF-M, and NF-L mRNAs encode intermediate filaments of ∼60, ∼100, and ∼110 kDa, respectively, but due to extensive post-translational modifications such as phosphorylation and glycosylation, NF-H, NF-M, and NF-L exhibit higher molecular weights after SDS-PAGE (Western) analysis of ∼68, ∼160, and ∼205 kDa, respectively; a dashed horizontal line at 1.0 is included for ease of comparison; N = 3 to 5 experiments for each treatment; ∗p < 0.01 (ANOVA); (E) Northern blot analysis – decreased NF-L in AD – Northern analysis of total NF-L mRNA in control (lanes 1–3) and AD (lanes 4–6) temporal lobe neocortex (Brodmann A22); the position of the migration of 28S and 18S RNA (4.7 and 1.9 knt, respectively) are marked on the right of the gel (upper panel); the size of the two prominent NF-L mRNA bands detected are respectively about 4.3 and 2.6 knt in length; an 18S RNA was used as an internal control marker (lower panel); (F) Northern blots were quantified in bar graph format showing the mean and one standard deviation of decreased NF-L mRNA signals in AD neocortex versus age-matched controls; in AD the 2 NF-L bands [between the 28S and 18S RNA markers of part (E)] together are about 0.3- to 0.4-fold AD over control; ∗p < 0.01 (ANOVA).
Fig 2: Decreased NF-L protein in LPS-treated HNG cells and in AD: ELISA and Western analysis. (A) results of sandwich ELISA analysis for NF-L protein in LPS-treated HNG cells using Abbexa (abx250460; Cambridge, United Kingdom) and/or LifeSpan BioSciences (LSBio; LS-F6701; Seattle WA, United States); the 68 kDa NF-L species is a particularly abundant intermediate filament protein, however in the presence of LPS the abundance of NF-L protein was found to be reduced to about 0.3-fold of control; a dashed horizontal line at 100 is included for ease of comparison; N = 3 to 5 experiments per determination; *p < 0.01 (ANOVA); (B) Western analysis of total NF-L protein (MW ~68 kDa) in control (pool of five controls and five AD temporal lobe neocortex Brodman A22) and total NF-L protein in control and LPS-treated HNG cells (at 2 weeks of culture; see Figure 4A); ß-actin protein (MW ~42 kDa) was used as an internal control marker in the same sample for each determination; (C) Western blots were quantified in bar graph format of decreased NF-L protein abundance in AD neocortex versus age-matched controls and in LPS-treated HNG cells versus age-matched controls; a dashed horizontal line at 1.0 is included for ease of comparison; the results of decreased NF-L expression for AD over control or LPS-treated HNG cells over control are highly significant; N = 3 to 5 experiments; *p < 0.01 (ANOVA).
Fig 3: Microbiome-derived LPS-mediated impairment of NF-L expression may contribute to atrophy of neurons and cytoskeletal disorganization that is characteristic of sporadic AD – the human GI-tract microbiome secretes a remarkably heterogeneous and complex mixture of neurotoxins including different varieties of lipooligosaccahrides (LOS), lipopolysaccharide (LPS), amyloids, small non-coding RNAs (sncRNAs) and exotoxins; recently several laboratories have provided evidence that these neurotoxins may transit GI-tract and blood-brain barriers and are present in the CNS and within aged or AD brain tissues; whether these microbiome-derived neurotoxins originate from the gastrointestinal (GI) tract microbiome, a possible brain microbiome or some dormant pathological microbiome is currently not well understood. Recent studies further suggest that the co-localization of pro-inflammatory LPS with AD-affected neuronal nuclei provides evidence that there may be a contribution of LPS to genotoxic events that support deficits in homeostatic gene expression that drive progressive AD-type change and provide support for pro-inflammatory neurodegeneration. This communication provides evidence that in both LPS-enveloped neuronal nuclei in AD neocortex and LPS-treated HNG cells in primary co-culture that there is a significant deficit in the expression of the neurofilament light-chain (NF-L), a neuron-specific cytoskeletal element known to be important in maintaining the shape and synaptic integrity of the neuron; see text and Figures 1–5 for additional details.
Supplier Page from Abbexa Ltd for Human Neurofilament, Light Polypeptide (NEFL) ELISA Kit