Fig 1: Affinity of LPS for neuronal nuclei in HNG cells and in AD brain. (A) Western analysis of lipopolysaccharide (LPS; ~37 kDa) signals in human brain temporal lobe neocortex [N = 4 controls and 4 sporadic Alzheimer’s disease (AD) cases; quantified in (B)] and in the hippocampus (C) [N = 3 controls and N = 3 sporadic AD cases]; quantified in (D) compared against ß-actin (~42 kDa) abundance in the same samples [using anti-Escherichia coli LPSs (cat# ab35654; Abcam, Cambridge, UK) and anti-ß-actin (cat# 3700; Cell Signaling, Danvers, MA, USA)]. Methodologies for Western analysis were previously described [41,42,43,45,46,49]. * p < 0.05. Densitometric readings of immune-reactive bands were obtained using ImageQuantTL (GE Healthcare, Chicago IL, USA) [12,19,20,41,42,43]; control and AD tissues were age- and gender-matched; there were no significant differences in age (controls, 82.5 ± 8.1 years; AD, 81.3 ± 8.8 years), gender (all female), postmortem interval (PMI; for all tissues, 3.8 h or less), RNA quality or RNA yield between the two groups; in these samples, LPS abundance was found to be on average greater than seven-fold more abundant in AD than the in the control neocortex; LPSs were found to be on average greater than 21-fold more abundant in AD than in the control hippocampus; trends of LPS-nuclear association studies in male brain tissue samples were similar. In (B,D), a dashed horizontal line at 100 is included for ease of comparison. (E) Immunohistochemical analyses. Human neuronal–glial (HNG) cells (transplantation grade) in primary co-culture were used to study the dynamics of LPS association with neurons. Blue-stained spherical and oval bodies are DAPI-stained nuclei; image indicates the affinity of LPSs for the neuronal nuclear envelope (white arrowheads); LPSs (red; ?max = 690 nm); neuron-specific ß-tubulin III (ßTUBIII)-stained (green; ?max = 520 nm) and nuclei (blue; ?max = 470 nm)-stained HNG cells; white arrows indicate punctate and perinuclear clustering of LPSs and LPS affinity for neuron-specific ß-tubulin III and the nuclear membrane, as was previously reported [41,42,43,44,45,46,47,48,49]. The incubation of HNG cells with LPSs or BF-LPS gave similar results; in AD, about ~76% of all LPS signals were found to be associated with neuronal cell nuclei; the association of LPS with the major cellular repository for genetic material suggests that the significance of this association may be genetic [41,42,43,44,45,46,47] (adapted from Figure 1B in reference [32]; bottom-right scale bar = 10 µm). (F) Immunohistochemical staining. Association and envelopment of AD-affected neocortical neuronal nuclei by LPS (red stain; ?max = 690 nm), DAPI (blue nuclear stain; ?max = 470 nm) and NeuN (neuron-specific green stain; ?max = 520 nm); the 3 white arrowheads indicate perinuclear ‘wrapping’ of LPS around neuronal nuclei. This feature has implications for reducing the transcriptional output of neurons [29,50] (microphotograph of sectioned tissue from human superior temporal lobe AD neocortex (Brodmann A22); figure adapted from Figure 3 in reference [52]; bottom-right scale bar = 20 µm).