Lipopolysaccharide (LPS) Accumulates in Neocortical Neurons of Alzheimer's Disease (AD) Brain and Impairs Transcription in Human Neuronal-Glial Primary Co-cultures

Abstract

Several independent laboratories have recently reported the detection of bacterial nucleic acid sequences or bacterial-derived neurotoxins, such as highly inflammatory lipopolysaccharide (LPS), within Alzheimer's disease (AD) affected brain tissues. Whether these bacterial neurotoxins originate from the gastrointestinal (GI) tract microbiome, a possible brain microbiome or some dormant pathological microbiome is currently not well understood. Previous studies indicate that the co-localization of pro-inflammatory LPS with AD-affected brain cell nuclei suggests that there may be a contribution of this neurotoxin to genotoxic events that support inflammatory neurodegeneration and failure in homeostatic gene expression. In this report we provide evidence that in sporadic AD, LPS progressively accumulates in neuronal parenchyma and appears to preferentially associate with the periphery of neuronal nuclei. Run-on transcription studies utilizing [α-³²P]-uridine triphosphate incorporation into newly synthesized total RNA further indicates that human neuronal-glial (HNG) cells in primary co-culture incubated with LPS exhibit significantly reduced output of DNA transcription products. These studies suggest that in AD LPS may impair the efficient readout of neuronal genetic information normally required for the homeostatic operation of brain cell function and may contribute to a progressive disruption in the read-out of genetic information.

Keywords: Alzheimer’s disease (AD); RNA Pol II transcription; inflammatory degeneration; lipopolysaccharide (LPS); run-on gene transcription.

Figure 1

Lipopolysaccharide (LPS) staining in the human superior temporal lobe neocortex (Brodmann A22) in control and Alzheimer’s disease (AD) brain; LPS (red stain; λ_max = 690 nm) and DAPI (blue stain; λ_max = 470 nm) staining of three control (Ct) (panels A–C) and three age-matched AD (panels D–F) temporal lobe neocortical tissues; the size of all microscope fields in this photo are equal; interestingly the slightly larger size of some DAPI-stained AD nuclei (blue) in panels (D–F) has been previously described in neuropathological studies of multiple anatomical regions in AD brain; the reason for this nuclear hypertrophy and plasticity in AD neuronal nuclei not entirely clear, but may be a “compensatory” mechanism requiring a more “expanded” euchromatin and increased transcriptional activity for some neurons that are attempting to repair neuronal and synaptic damage (Iacono et al., 2008); all panels magnification 63×; scale bar = 20 μm.

Figure 2

Association of LPS with the periphery of neuronal nuclei in AD neocortex—LPS (red stain; λ_max = 690 nm), DAPI (blue stain; λ_max = 470 nm) and NeuN (green stain; λ_max = 520 nm) staining of control and age-matched human superior temporal lobe AD neocortex (Brodmann A22); note that in the right-most AD panels (C–G,D,H) about 90% of all LPS signals were associated with NeuN (green-staining; neuronal) and DAPI (blue staining) nuclei; panels (A,B,E,F) are from control neocortex; panels (C,D,G,H) are from AD neocortex: quantitative analysis of LPS association with neuronal cells in bar graph format (panel I); LPS staining (red) was quantified as average percentage of neuronal area associated with neuronal cells (green); LPS staining (red) was subjected to co-localization analysis with the neuronal marker NeuN (green) and/or nuclear marker (blue); the highlighted co-localized area was then quantified as a percentage of neuronal area in the image; the analysis was performed by using NIH ImageJ software (see text for further details); data are presented as one mean ± one standard deviation (SD); *p < 0.05 vs. control; for all panels magnification 63×; scale bar = 20 μm.

Figure 3

Primary human neuronal-glial (HNG) cells after ~2 weeks in primary co-culture; the cell density is approximately 75% neurons and 25% astroglia at ~60% confluency; human primary neuronal and glial “support” cell co-cultures are utilized, because human neuronal cells do not culture well by themselves (Cui et al., 2010); neuronal cells are stained with neuron-specific β-tubulin (red; λ_max = 690 nm), glial cells are stained with glial-specific glial fibrillary acidic protein (GFAP; green; λ_max = 525 nm), and nuclei are stained with DAPI/Hoechst 33258 stain (blue; λ_max = 470 nm); photo magnification 30×; scale bar = 50 μm.