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. 2020;75(4):1361-1376.
doi: 10.3233/JAD-200393.

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

Ursula Haditsch  1 , Theresa Roth  1 , Leo Rodriguez  1 , Sandy Hancock  2 , Thomas Cecere  2 , Mai Nguyen  1 , Shirin Arastu-Kapur  1 , Sean Broce  1 , Debasish Raha  1 , Casey C Lynch  1 , Leslie J Holsinger  1 , Stephen S Dominy  1 , Florian Ermini  1
Affiliations

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

Ursula Haditsch et al. J Alzheimers Dis. 2020.

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 phospho-tau/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.

Keywords: Alzheimer’s disease; Porphyromonas gingivalis; gingipain cysteine endopeptidases; in vitro techniques; multivesicular bodies; synapses; tau protein.

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Conflict of interest statement

Authors’ disclosures are available online (https://www.j-alz.com/manuscript-disclosures/20-0393).

Figures

Fig.1
Fig.1
Viability of iPSC derived neurons and P. gingivalis for up to 72 h after infection. A) The number of P. gingivalis colocalized with neurons increases with increased numbers of P. gingivalis added to cultures. Kruskal-Wallis test with Dunn’s multiple comparison test: ****p < 0.001. B) Neuron cell death assay quantified by the ratio of dead cells over the total number of nuclei 24 h after infection with different BCRs. Kruskal-Wallis test with Dunn’s multiple comparison test: ****p < 0.0001. C) Neuron cell death assay 24 h, 48 h, and 72 h after infection with BCR 300. Two-way ANOVA with Tukey’s multiple comparison test: **p < 0.01, ***p < 0.001. D) Quantification of bacteria 24 h, 48 h, and 72 h after infection with BCR 300 by colony assay (CFU), qPCR, and high content screening (HCS). E) RT-qPCR analysis of neuron lysates for Kgp, Rgp mRNA, and P. gingivalis specific 16 S rRNA. A-C) Mean with 95% confidence intervals. D-E) Geometric mean with geometric standard deviation.
Fig.2
Fig.2
P. gingivalis is internalized and secretes active gingipains for up to 72 h. A) Maximal projection of a confocal image stack of P. gingivalis (magenta) infected neurons stained for the membrane specific protein Thy1 (cyan). a’) 3D reconstruction. a”) Same as a’ without the Thy1 channel. Yellow arrows point to aggregates of P. gingivalis partially engulfed in membrane. White arrows point at P. gingivalis completely internalized in the neuronal membrane. Scale bar 20μm. B) Maximal projection of confocal image stack immunofluorescence stained for Rgp (green), P. gingivalis (red), and MAP2 (blue): secreted Rgp creates a field around neuron associated bacterial clusters. b-b””) 3D iso-surface reconstruction of the area indicated in B. b) MAP2 and Rgp channels omitted. b”) MAP2 channel omitted. b”’) Rgp channel omitted. b””) All channels visible. Gel electrophoresis of cell lysates incubated with a Cy-5 conjugated activity probe for Rgp (C) and Kgp (D). Arrows indicate the expected protein sizes at 45 kDa and 70 kDa for Rgp (C) and 50 kDa for Kgp (D). E and F are the corresponding western blots to the gels depicted in C and D, respectively. Blots were stained with antibodies against Rgp (E) and Kgp (F). Full length gels and western blots are presented in Supplementary Figure 5. G) Colorimetric activity assay for Rgp activity from cell lysates at 24 h, 48 h, and 72 h after infection.
Fig.3
Fig.3
P. gingivalis attaches to neurons, is internalized, and is associated with disruption of the cytoskeleton. A,B) Examples of extracellular P. gingivalis contacting a neurite and inducing a membrane swelling resembling an actin pedestal (arrow). This was associated with an electron dense band along the contact surface to P. gingivalis (arrowhead). C) Some bacteria were internalized by membrane swellings on neurites (arrow). D) An example of two P. gingivalis organisms (circle arrows) residing within a neurite without being bound by a distinct surrounding membrane and one P. gingivalis inside a membrane (arrow). E) Two P. gingivalis within the cell body that are non-membrane bound (circle arrows) and are surrounded by heterogenous electron dense matrix (arrowheads) with a peripheral zone of neurofilaments and microtubules (double headed arrows).
Fig.4
Fig.4
Intraneuronal P. gingivalis is found in the endosomal/lysosomal pathway. A) Two groups of P. gingivalis (white rectangles) within membrane bound structures resembling lysosomes. a’) Enlarged inset of A (lower square) showing an aggregate of two intact bacteria (arrows) and a partially degraded bacterium a”) Enlarged inset of A (upper square) a pair of intact bacteria (arrows) surrounded by an accumulation of lipid droplets (stars). B) Two P. gingivalis inside a single membrane (arrow), the surrounding cytosol contains rough endoplasmic reticulum and neurofilament looks unperturbed except for a small electron lucent halo surrounding the bacteria containing compartment. Percentage of internalized P. gingivalis colocalizing with early endosomes (C), late endosomes (D), and lysosomes (E). Mean with 95% confidence interval. Student’s t-test, ***p < 0.005. F) Live imaging of P. gingivalis (red), lysotracker (green), and NeuO (blue). Line scans in a neurite (inset 1) and the cell body (inset 2) confirm colocalization of P. gingivalis with migrating lysosomes over time. In inset 1, both bacteria continue to colocalize with lysotracker (arrowheads). In inset 2, three bacteria colocalize at time 0. At 21 min, one remains in the lysosome (arrowhead), one has moved away from the line scan (small arrow), and one is not colocalizing with lysotracker anymore (arrow).
Fig.5
Fig.5
Infection with P. gingivalis causes an accumulation of autophagosomes and multivesicular bodies. A) Accumulation of multivesicular bodies (arrow heads) in the perikaryon of neuron infected with P. gingivalis. B) Magnified inset of A. C,D) Two examples of variably sized membrane bound vesicles containing heterogeneous amorphous electron dense material interpreted as secondary lysosomes (arrowheads) or occasional concentric lamellar bodies interpreted as autophagosomes (arrows). Quantification of mean neurite thickness (E) and mean neurite area (F) showed significant increases in infected neuron cultures. Mean with 95% confidence interval; **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig.6
Fig.6
P. gingivalis degrades tau, increases the ratio of ptau/tau and decreases synapse density. A) Western blots stained with total tau and synapsin antibodies for infected and noninfected lysates at 24 h and 48 h after infection. GAPDH was included as a loading control. Full length western blots are presented in Supplementary Figure 6. B,C) Densitometry of western blots depicted in A normalized to GAPDH. Mean with 95% confidence interval, 1-way ANOVA with Tukey’s multiple comparison test ****p < 0.0001, ***p < 0.001. Multiplex ELISA quantification of ptau(T231) (D), total tau (E), and the calculated ptau(T231)/total tau ratio (F). Mean with 95% confidence interval, 1-way ANOVA with Tukey’s multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001. G) Multichannel maximum projection of a confocal stack with synaptophysin (green), P. gingivalis (red), and a universal membrane marker (blue) used as a cell mask for high content screening. The blue channel has been omitted in the right-side panel (g). Arrows point at P. gingivalis associated with neurons; arrow heads point at synaptophysin positive synaptic buttons. Scale bar 50μm. H) Quantification of synaptic buttons 24 h and 48 h after infection. Mean with 95% confidence interval, 2-way ANOVA with Dunnett’s multiple comparison test: **p < 0.01, ***p < 0.005.
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