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.

Background We identified the bacterial pathogen, Porphyromonas gingivalis (Pg), and its protease virulence factors, gingipains, in the brain of patients with Alzheimer’s disease (AD). Gingipain levels in AD brains were shown to significantly correlate with AD diagnosis and tau and ubiquitin pathology. The neurodegeneration and AD‐like pathology in Pg infected mice were blocked after oral administration of COR388, an orally bioavailable, brain penetrant small‐molecule that irreversibly inhibits lysine‐gingipain. Since ApoE4 is the greatest genetic risk factor for sporadic AD, we investigated if ApoE proteins are targets of gingipain proteolysis. Method In vitro proteolysis of recombinant ApoE proteins, Mass Spectrometry (MS) analysis of gingipain cleavage sites, and detection of ApoE proteolytic fragments in brain and CSF. Result ApoE proteins were cleaved by gingipains rapidly, and ApoE4 was a preferred substrate over ApoE3. Gingipain inhibitors blocked ApoE proteolysis. MS analysis confirmed higher susceptibility of ApoE4 to gingipain cleavage. MS analysis enabled the identification of cleavage sites and the majority of these sites were concentrated near the carboxy‐terminal of the protein. MS analysis performed on low‐molecular‐weight (LMW) fragments of ApoE4 from a human AD brain, homozygous for the APOE4 allele, identified peptide fragments from this same region. Western blot analysis of AD CSF revealed a higher level of LMW ApoE proteolytic cleavage products in AD CSF than non‐AD CSF. A significant decrease in LMW ApoE fragments in CSF was detected in AD subjects following 28 days of administration of COR388 compared to placebo in a Phase1b clinical study. Conclusion Our data suggest that gingipain proteases may mediate ApoE proteolysis in the brain, with higher susceptibility of ApoE4, providing a link between P. gingivalis infection and the APOE4 allele, a major risk factor in AD. Based on the literature, fragmentation of ApoE4 by gingipains would be likely to reduce synaptic maintenance and regulation of immune response as well as aggravate toxicity to cells through the generation of fragments. COR388, a small‐molecule inhibitor of lysine‐gingipain, is currently being tested in a large Phase 2/3 clinical study in subjects with mild‐to‐moderate AD.

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Background Porphyromonas gingivalis and its proteolytic virulence factors lysine‐gingipain (Kgp) and arginine‐gingipain (Rgp) are emerging as major etiologic agents in the pathogenesis of Alzheimer’s disease (AD). We have previously reported that gingipains colocalize with neuronal cell bodies in over 90% of AD brains studied and the presence of gingipains strongly correlates with tau pathology. We have found tau protein to be a substrate for both Kgp and Rgp and have identified over forty cleavage sites combined. Here we used iPSC‐derived neuron cultures and neuron‐astrocyte‐microglia co‐cultures to investigate the neuropathological mechanisms resulting from persistent infection with P. gingivalis and gingipain exposure and highlight the therapeutic potential of gingipain inhibitors. Method IPSC derived neuron cultures and neuron‐astrocyte‐microglia co‐cultures were infected with P. gingivalis and analyzed by high content screening (HCS), confocal microscopy, transmission electron microscopy (TEM), protein and RNA expression analysis. Result Intracellular P. gingivalis bacteria were observed in neurons for 3 days or longer after infection. Infection produced significant synaptic loss that was completely prevented by Kgp and Rgp inhibitors including COR388. Using electron microscopy, we found an accumulation of autophagic vacuoles in dystrophic neurites reminiscent of ultrastructural findings in AD brains. Furthermore, gingipain exposure resulted in acute fragmentation of tau and increased tau phosphorylation, resulting in destabilized microtubules and disrupted vesicle transport. Transport disruption by gingipains accounted for the accumulation of autophagic vacuoles in dystrophic neurites, as no dystrophic neurites were observed in cultures treated with gingipain inhibitors. When co‐cultures of microglia, astrocytes and neurons were infected, activated microglia displayed the highest number of internalized bacteria and responded to infection by upregulation of inflammatory markers associated with AD pathology. Conclusion We demonstrate in an in vitro model system that P. gingivalis infection can result in a neurodegenerative phenotype consistent with pathology found in Alzheimer’s disease. With this model we show that exposure of neurons to P. gingivalis infection results in synaptic loss and dystrophic neurites which can be prevented by gingipain inhibitors.

Background We recently demonstrated the presence of the bacterial pathogen, Porphyromonas gingivalis (Pg), and its protease virulence factors, known as gingipains, in greater than 90% of AD brains. Brain gingipain levels significantly correlated with AD diagnosis and tau and ubiquitin pathology. Mechanistic studies in wild type mice demonstrated that Pg invades the brain after infection of the oral cavity, resulting in the development of neuropathology consistent with that of AD. These effects were blocked in mice after oral administration of the gingipain inhibitor COR388. In addition to neuropathology, AD is often accompanied by the comorbidity of cardiovascular disease. Pg has been linked to the development of atherosclerosis and the bacteria are found abundant within atheromatous plaque. We investigated if COR388 was efficacious in treating Pg‐accelerated atherosclerosis in a rabbit model. Method Eighteen New Zealand White rabbits on a 13‐week regimen of 0.5% cholesterol diet to induce atherosclerosis were randomized in the study. Twelve rabbits were orally infected with Pg 3x/week for 6 weeks for oral disease induction and 6 rabbits were sham‐infected. Six Pg –infected rabbits were orally administered COR388 10mg/kg once daily for the entire 13 weeks of the study (prevention protocol), and 6 Pg‐infected rabbits received vehicle for 13 weeks. Six sham‐infected rabbits received vehicle for 13 weeks. At 13 weeks, atherosclerotic plaques were quantified by assessing Sudan IV staining, histology, and ex vivo MRI. Serum levels of C‐reactive protein (CRP) were evaluated as a measure of systemic inflammation. Result Pg‐infected rabbits exhibited accelerated atherosclerotic disease severity than sham‐infected animals. COR388 diminished this atherogenesis compared to vehicle controls as evidenced by significantly less arterial plaque and a lower intima/media ratio. COR388 treatment also significantly reduced systemic levels of CRP. Conclusion COR388, a novel lysine‐gingipain inhibitor, is currently being tested in a Phase 2/3 clinical trial to target Pg for the treatment of AD. Based on the current preclinical data reported here, COR388 has the potential to attenuate atheroma formation and systemic inflammation in Pg‐ induced atherosclerosis and therefore may have beneficial effects on comorbid cardiovascular disease in Pg‐associated AD.