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

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.

Female-Specific Role of Progranulin to Suppress Bone Formation.

Progranulin (PGRN) is best known as a glial protein for which deficiency leads to the most common inherited form of frontotemporal dementia. Recently, PGRN has been found to be an adipokine associated with diet-induced obesity and insulin resistance. Therefore, PGRN may have homeostatic effects on bone because PGRN is reported to promote the differentiation of bone-resorbing osteoclasts. We investigated the actions of PGRN on bone using PGRN gene (Grn) knockout (KO) mice and transgenic mice with PGRN mutation and surprisingly found that loss of PGRN prevented the bone loss in female mice induced by aging and estrogen deficiency, whereas it had no effect on male bones during aging. Strikingly, bone formation was increased in female (but not male) PGRN KO mice. We also found that loss of PGRN inhibited bone resorption and osteoclastogenesis in both male and female mice and promoted the production of osteogenic factors in osteoclast lineage cells. These results indicate that PGRN serves to uncouple bone turnover in female mice by promoting bone resorption and suppressing bone formation. Furthermore, we demonstrated that microglial cells/macrophages, but not adipocytes, are an important source of PGRN in producing negative skeletal effects in females. Targeting PGRN production by microglial cells/macrophage-lineage cells may provide a therapeutic approach for the treatment of osteoporosis in females.

Sirtuin-3 Promotes Adipogenesis, Osteoclastogenesis, and Bone Loss in Aging Male Mice.

Sirtuin-3 (Sirt3) is an essential metabolic regulatory enzyme that plays an important role in mitochondrial metabolism, but its role in bone marrow and skeletal homeostasis remains largely unknown. In this study, we hypothesize that increased expression of Sirt3 plays a role in skeletal aging. Using mice that overexpress Sirt3 [i.e., Sirt3 transgenic (Sirt3Tg)], we show that Sirt3 is a positive regulator of adipogenesis and osteoclastogenesis and a negative regulator of skeletal homeostasis. Sirt3Tg mice exhibited more adipocytes in the tibia compared with control mice. Bone marrow stromal cells (BMSCs) from Sirt3Tg mice displayed an enhanced ability to differentiate into adipocytes compared with control BMSCs. We found a 2.5-fold increase in the number of osteoclasts on the bone surface in Sirt3Tg mice compared with control mice (P < 0.03), and increased osteoclastogenesis in vitro. Importantly, Sirt3 activates the mechanistic target of rapamycin (mTOR) pathway to regulate osteoclastogenesis. Sirt3Tg male mice exhibited a significant reduction in cortical thickness at the tibiofibular junction (P < 0.05). In summary, Sirt3 activity in bone marrow cells is associated with increased adipogenesis, increased osteoclastogenesis through activation of mTOR signaling, and reduced bone mass. Interestingly, Sirt3 expression in bone marrow cells increases during aging, suggesting that Sirt3 promotes age-related adipogenesis and osteoclastogenesis associated with bone loss. These findings identify Sirt3 as an important regulator of adipogenesis and skeletal homeostasis in vivo and identify Sirt3 as a potential target for the treatment of osteoporosis.

Osteoblast-derived FGF9 regulates skeletal homeostasis.

FGF9 has complex and important roles in skeletal development and repair. We have previously observed that Fgf9 expression in osteoblasts (OBs) is regulated by G protein signaling and therefore the present study was done to determine whether OB-derived FGF9 was important in skeletal homeostasis. To directly test this idea, we deleted functional expression of Fgf9 gene in OBs using a 2.3kb collagen type I promoter-driven Cre transgenic mouse line (Fgf9OB-/-). Both Fgf9 knockout (Fgf9OB-/-) and the Fgf9 floxed littermates (Fgf9fl/fl) mice were fully backcrossed and maintained in an FBV/N background. Three month old Fgf9OB-/- mice displayed a significant decrease in cancellous bone and bone formation in the distal femur and a significant decrease in cortical thickness at the TFJ. Strikingly, female Fgf9OB-/- mice did not display altered bone mass. Continuous treatment of mouse BMSCs with exogenous FGF9 inhibited mouse BMSC mineralization while acute treatment increased the proliferation of progenitors, an effect requiring the activation of Akt1. Our results suggest that mature OBs are an important source of FGF9, positively regulating skeletal homeostasis in male mice. Osteoblast-derived FGF9 may serve a paracrine role to maintain the osteogenic progenitor cell population through activation of Akt signaling.

Negative Skeletal Effects of Locally Produced Adiponectin.

Epidemiological studies show that high circulating levels of adiponectin are associated with low bone mineral density. The effect of adiponectin on skeletal homeostasis, on osteoblasts in particular, remains controversial. We investigated this issue using mice with adipocyte-specific over-expression of adiponectin (AdTg). MicroCT and histomorphometric analysis revealed decreases (15%) in fractional bone volume in AdTg mice at the proximal tibia with no changes at the distal femur. Cortical bone thickness at mid-shafts of the tibia and at the tibiofibular junction was reduced (3-4%) in AdTg mice. Dynamic histomorphometry at the proximal tibia in AdTg mice revealed inhibition of bone formation. AdTg mice had increased numbers of adipocytes in close proximity to trabecular bone in the tibia, associated with increased adiponectin levels in tibial marrow. Treatment of BMSCs with adiponectin after initiation of osteoblastic differentiation resulted in reduced mineralized colony formation and reduced expression of mRNA of osteoblastic genes, osterix (70%), Runx2 (52%), alkaline phosphatase (72%), Col1 (74%), and osteocalcin (81%). Adiponectin treatment of differentiating osteoblasts increased expression of the osteoblast genes PPARγ (32%) and C/ebpα (55%) and increased adipocyte colony formation. These data suggest a model in which locally produced adiponectin plays a negative role in regulating skeletal homeostasis through inhibition of bone formation and by promoting an adipogenic phenotype.

Effects of blockade of endogenous Gi signaling in Tie2-expressing cells on bone formation in a mouse model of heterotopic ossification.

Available evidence indicates that some Tie2-expressing (Tie2(+) ) cells serve as multipotent progenitors that have robust BMP-dependent osteogenic activity and mediate heterotopic ossification (HO). Since signaling through the G protein Gi is required for cell motility, we hypothesized that blockade of endogenous Gi signaling in Tie2(+) cell populations would prevent HO formation. Blockade of Gi signaling in Tie2(+) cells was accomplished in transgenic mice with expression of pertussis toxin (PTX) under the control of the Tie2 promoter (Tie2(+) /PTX(+) ). Bone formation within HOs was evaluated 2 weeks after BMP injection. Expression of PTX in Tie2(+) cells significantly reduced the bone volume (BV) of HOs in male and female mice. Orthotopic bones were assessed at the distal femur and expression of PTX significantly increased trabecular bone fractional volume and bone formation rate in females only. In adult Tie2(+) /GFP(+) mice, GFP(+) cells appeared both inside and at the surfaces of bone tissue within HOs and in orthotopic bones. In summary, blockade of Gi signaling in Tie2(+) cells reduced the accrual of HOs and stimulated osteogenesis in orthotopic bones. Targeting of Gi protein coupled receptors in Tie2(+) cells may be a novel therapeutic strategy in states of abnormal bone formation such as osteoporosis and HO.