Porphyromonas gingivalis-Lipopolysaccharide Induced Caspase-4 Dependent Noncanonical Inflammasome Activation Drives Alzheimer's Disease Pathologies

Abstract

Chronic periodontitis, driven by the keystone pathogen Porphyromonas gingivalis, has been increasingly associated with Alzheimer's disease (AD) and AD-related dementias (ADRDs). However, the mechanisms through which P. gingivalis-lipopolysaccharide (LPS)-induced release of neuroinflammatory proteins contribute to the pathogenesis of AD and ADRD remain inadequately understood. Caspase-4, a critical mediator of neuroinflammation, plays a pivotal role in these processes following exposure to P. gingivalis-LPS. In this study, we investigated the mechanistic role of caspase-4 in P. gingivalis-LPS-induced IL-1β production, neuroinflammation, oxidative stress, and mitochondrial alterations in human neuronal and microglial cell lines. Silencing of caspase-4 significantly attenuated IL-1β secretion by inhibiting the activation of the caspase-4-NLRP3-caspase-1-gasdermin D inflammasome pathway, confirming its role in neuroinflammation. Moreover, caspase-4 silencing reduced the activation of amyloid precursor protein and presenilin-1, as well as the secretion of amyloid-β peptides, suggesting a role for caspase-4 in amyloidogenesis. Caspase-4 inhibition also restored the expression of key neuroinflammatory markers, such as total tau, VEGF, TGF, and IL-6, highlighting its central role in regulating neuroinflammatory processes. Furthermore, caspase-4 modulated oxidative stress by regulating reactive oxygen species production and reducing oxidative stress markers like inducible nitric oxide synthase and 4-hydroxynonenal. Additionally, caspase-4 influenced mitochondrial membrane potential, mitochondrial biogenesis, fission, fusion, mitochondrial respiration, and ATP production, all of which were impaired by P. gingivalis-LPS but restored with caspase-4 inhibition. These findings provide novel insights into the role of caspase-4 in P. gingivalis-LPS-induced neuroinflammation, oxidative stress, and mitochondrial dysfunction, demonstrating caspase-4 as a potential therapeutic target for neurodegenerative conditions associated with AD and related dementias.

Keywords: Porphyromonas gingivalis; caspase-4; lipopolysaccharide; mitochondrial dysfunction; neuroinflammation; oxidative stress.

Figure 2

P. gingivalis-LPS upregulates Alzheimer’s disease-associated proteins APP, PS1, reelin, and Aβ in a caspase-4 dependent manner in SH-SY5Y cells. (A) Representative Western blots demonstrate that silencing of caspase-4 using siRNA restores the protein expression of APP, PS1, and reelin following P. gingivalis-LPS treatment in SH-SY5Y cells (n = 3). β-Actin was used as a loading control. (B–D) Quantification of relative protein levels of APP, PS1, and reelin normalized to β-Actin is shown in the graphs. (E,F) ELISA analysis of Aβ_1–42 and Aβ_1–40 peptides in SH-SY5Y cells following P. gingivalis-LPS treatment, in combination with caspase-4 siRNA (n = 4). (G,H) ELISA analysis of Aβ_1–42 and Aβ_1–40 peptides following P. gingivalis-LPS treatment and caspase-4 inhibition with Ac-LEVD-CHO (n = 4). Data are expressed as mean ± SEM and represent at least three independent experiments. Statistical significance is indicated as follows: **** p < 0.0001 as determined by one-way ANOVA with Tukey’s multiple comparisons test.

Figure 1

P. gingivalis-LPS induces IL-1β secretion via a caspase-4 dependent non-canonical inflammasome pathway. SH-SY5Y cells were pretreated with Ac-LEVD-CHO (a caspase-4 inhibitor) for 1 h before LPS treatment. (A) After 24 h, culture supernatants were collected, and IL-1β secretion was quantified by ELISA (n = 4). (B) Representative Western blots depict the upregulation of caspase-4, NLRP3, caspase-1, GSDMD, GSDMD-N, and IL-1β in response to P. gingivalis-LPS, along with the reversal of this effect following caspase-4 silencing via siRNA. β-Actin was used as a loading control. (C–H) Quantification of relative protein expression of caspase-4, NLRP3, caspase-1, GSDMD, GSDMD-N, and IL-1β, normalized to β-Actin, is presented in the graphs (n = 3). Data are expressed as mean ± SEM and represent at least three independent experiments. Statistical significance is indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, determined by one-way ANOVA with Tukey’s multiple comparisons test.

Figure 3

P. gingivalis-LPS induces neuroinflammatory markers via caspase-4 activation in SH-SY5Y cells. Relative mRNA expression of neuroinflammatory markers, including (A) T-Tau (Total Tau), (B) VEGF, (C) TGF-β, (D) TNF-α, and (E) IL-6, was significantly increased following P. gingivalis-LPS treatment and reversed by caspase-4 silencing using siRNA (n = 3). (F,G) ELISA analysis of phosphorylated tau at T181 and T217 in response to P. gingivalis-LPS treatment along with caspase-4 siRNA, and a caspase-4 inhibitor, Ac-LEVD-CHO (n = 4). Data are expressed as mean ± SEM and represent at least three independent experiments. Statistical significance is indicated as follows: ** p < 0.01; *** p < 0.001; **** p < 0.0001 determined by one-way ANOVA with Tukey’s multiple comparisons test.

Figure 4

P. gingivalis-LPS induces oxidative stress and disrupts mitochondrial membrane potential via caspase-4 dependent inflammasome activation. SH-SY5Y cells were pretreated with Ac-LEVD-CHO for 1 h before LPS treatment. (A) ROS-producing cells were analyzed by flow cytometry using MitoSOX Red. (B) Bar graph showing a significant increase in the percentage of MitoSOX-positive cells upon LPS treatment, which was significantly reversed with Ac-LEVD-CHO. (C) Mitochondrial membrane potential was assessed using JC-1 staining. (D) The graph shows the ratio of JC-1 aggregates (red) to monomers (green), which was significantly decreased following LPS treatment and restored upon caspase-4 inhibition with Ac-LEVD-CHO. (E) Representative Western blots display the upregulation of iNOS and 4-HNE, along with the downregulation of MnSOD in response to P. gingivalis-LPS, with significant recovery of these markers following caspase-4 silencing via siRNA (n = 3). β-Actin was used as a loading control. (F–H) Quantification of relative protein expression of iNOS, 4-HNE, and MnSOD, normalized to β-Actin, is presented in the graphs (n = 3). Data are expressed as mean ± SEM and represent at least three independent experiments. Statistical significance is indicated as follows: **** p < 0.0001, determined by one-way ANOVA with Tukey’s multiple comparisons test.

Figure 5

P. gingivalis-LPS inhibits mitochondrial biogenesis, fission, and fusion through caspase-4 activation. (A–D) RT-qPCR analysis of mitochondrial biogenesis markers, including PGC-1α, PGC-1β, NRF, and TFAM, revealed a significant downregulation of their mRNA expression following P. gingivalis-LPS treatment, which was significantly restored by caspase-4 inhibition using Ac-LEVD-CHO (n = 4). (E) Representative Western blots show the downregulation of PGC-1α, NT-PGC-1α, and PGC-1β in response to P. gingivalis-LPS, with recovery observed following caspase-4 silencing via siRNA. (F–H) Quantification of relative protein expression of PGC-1α, NT-PGC-1α, and PGC-1β, normalized to β-Actin, is presented in the graphs (n = 3). (I,J) RT-qPCR analysis of mitochondrial pro-fission (Fis1, Drp1) and (K–M) pro-fusion (Mfn1, Mfn2, Opa1) markers showed a significant decrease in their mRNA expression following LPS treatment, which was rescued by Ac-LEVD-CHO (n = 4). Data are expressed as mean ± SEM and represent at least three independent experiments. Statistical significance is indicated as follows: ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: p > 0.05, determined by one-way ANOVA with Tukey’s multiple comparisons test.

Figure 6

Mitochondrial functional analysis of OCR, ECAR, and total ATP production in SH-SY5Y cells. Cells were transfected with caspase-4 siRNA for 24 h before P. gingivalis-LPS treatment. (A) Seahorse XF Cell Mito Stress Test results showing mean ± SEM normalized to an equal number of cells. Oligomycin, FCCP, and rotenone were sequentially injected to assess mitochondrial ATP production, maximal respiration, and non-mitochondrial respiration. (B) Quantification of basal respiration, maximal respiration, and spare respiratory capacity revealed a significant increase following LPS treatment, which was reversed by caspase-4 siRNA. (C) Glycolytic rate assay data presented as mean ± SEM and normalized to equal cell numbers. Rotenone/antimycin A was injected to inhibit mitochondrial function, and 2-DG was used to block glycolysis as an internal control. (D) Graphs show the calculated glycolytic parameters, including basal and compensatory glycolysis. Compensatory glycolysis was significantly increased following LPS treatment and restored by caspase-4 siRNA. (E) Real-time ATP rate assay indicated a significant reduction in total ATP production from both oxidative phosphorylation and glycolysis following LPS treatment, which recovered upon caspase-4 silencing (n = 10). Data are expressed as mean ± SEM, representing at least three independent experiments. Statistical significance is indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, determined by two-way ANOVA with Tukey’s multiple comparisons test.

Figure 7

Functional analysis of mitochondrial respiratory chain activity in P. gingivalis-LPS transfected SH-SY5Y cells. Cells were treated with P. gingivalis-LPS with or without the caspase-4 inhibitor Ac-LEVD-CHO, then permeabilized with digitonin to assess the oxygen consumption rate (OCR) at various electron transport chain complexes (n = 3). OCR is shown as a function of time, with blue lines indicating the time points of substrate and inhibitor injections. (A) Representative trace from high-resolution respirometry using a multiple substrate-inhibitor titration protocol. The protocol includes malate/glutamate (complex I substrates), ADP (OXPHOS capacity), rotenone (complex I inhibition), succinate (complex II substrate), antimycin A (complex III inhibition), ascorbate/TMPD (complex IV), and sodium azide (complex IV inhibition). Oxygen concentration is depicted by blue lines, and respiration rate is represented by red lines. (B) Quantification of OCR at complexes I, II, and IV shows significantly increased OCR in P. gingivalis-LPS treated cells, which was reversed by Ac-LEVD-CHO treatment. (C) Representative Western blot analysis of OXPHOS mitochondrial complexes in SH-SY5Y cells treated with P. gingivalis-LPS, with or without caspase-4 siRNA using cocktail antibody against complexes I–V. (D–G) Relative quantification of protein levels of complex I, II, III, and V, normalized to β-Actin, shows a significant increase in complex I and II protein expression following P. gingivalis-LPS treatment, which was reversed by caspase-4 silencing. No significant change in complex III and complex V protein expression was observed between control and P. gingivalis-LPS treated groups. Data are expressed as mean ± SEM, representing at least three independent experiments. Statistical significance is indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: p > 0.05, determined by two-way ANOVA with Tukey’s multiple comparisons test.