Alzheimer’s Disease-Like Neurodegeneration in Porphyromonas gingivalis Infected Neurons with Persistent Expression of Active Gingipains

Abstract

Background: Porphyromonas gingivalis (P. gingivalis) and its gingipain virulence factors have been identified as pathogenic effectors in Alzheimer's disease (AD). In a recent study we demonstrated the presence of gingipains in over 90% of postmortem AD brains, with gingipains localizing to the cytoplasm of neurons. However, infection of neurons by P. gingivalis has not been previously reported. Objective: To demonstrate intraneuronal P. gingivalis and gingipain expression in vitro after infecting neurons derived from human inducible pluripotent stem cells (iPSC) with P. gingivalis for 24, 48, and 72 h. Methods: Infection was characterized by transmission electron microscopy, confocal microscopy, and bacterial colony forming unit assays. Gingipain expression was monitored by immunofluorescence and RT-qPCR, and protease activity monitored with activity-based probes. Neurodegenerative endpoints were assessed by immunofluorescence, western blot, and ELISA. Results: Neurons survived the initial infection and showed time dependent, infection induced cell death. P. gingivalis was found free in the cytoplasm or in lysosomes. Infected neurons displayed an accumulation of autophagic vacuoles and multivesicular bodies. Tau protein was strongly degraded, and phosphorylation increased at T231. Over time, the density of presynaptic boutons was decreased. Conclusion: P. gingivalis can invade and persist in mature neurons. Infected neurons display signs of AD-like neuropathology including the accumulation of autophagic vacuoles and multivesicular bodies, cytoskeleton disruption, an increase in phosphotau/tau ratio, and synapse loss. Infection of iPSC-derived mature neurons by P. gingivalis provides a novel model system to study the cellular mechanisms leading to AD and to investigate the potential of new therapeutic approaches.

Interaction between genetic factors, Porphyromonas gingivalis and microglia to promote Alzheimer’s disease

“…Ilievski et al [8] have proven the concept that chronic periodontal infection can result in the formation of the diagnostic neuropathology lesions consistent with AD. Haditsch et al [9] confirmed the findings of Ilievski et al [8] for p-Tau on Ser396 and additionally demonstrated an increased tau phosphorylation at Thr231 following P. gingivalis infection with persistent gingipain expression with ongoing neurodegeneration.…”

“…As mentioned, Haditsch et al [9] reported AD-like neurodegeneration in P. gingivalis infected neurons in an in-vitro culture system with persistent expression of active gingipains. Following infection with live P. gingivalis (ATCC 33277) 25% of the neurons were lost in a time-dependent manner.…”

Alzheimer’s Disease-Like Pathology Triggered by Porphyromonas gingivalis in Wild Type Rats Is Serotype Dependent

“…It was recently reported that gingipain inhibition reduces the bacterial load of an established P. gingivalis brain infection, decreases neuroinflammation, and rescues neurons in the hippocampus, revealing that this endopeptidase has a pivotal role in the mechanism of brain damage induced by P. gingivalis (34). Gingipains are likely to participate in the process of neuronal tissue invasion (38).…”

“…Interestingly, the possibility that the immune response induced in the brain might differ depending on the infecting capsular P. gingivalis serotype has never been exploited. Microglia are known to discriminate against different bacterial antigens, triggering the activation of divergent pathways according to the antigens recognized ( 35 – 38 ). Thus, it is possible that some P. gingivalis strains might be more virulent than others in causing Alzheimer´s like-disease.…”

Susceptibility to gingipains and transcriptomic response toP. gingivalishighlights the ribosome, hypothalamus, and cholinergic neurons

“…These are independent signals as only two genes are in both of these sets. A recent study by Haditsch et al, found that neurons derived from human inducible pluripotent stem cells, when infected with P. gingivalis , had a significant loss of synapse density (Haditsch et al 2020a) . This loss was more pronounced than neuron cell death.…”