The Alzheimer disease protective mutation A2T modulates kinetic and thermodynamic properties of amyloid-ß (Aß) aggregation.

Missense mutations in alanine 673 of the amyloid precursor protein (APP), which corresponds to the second alanine of the amyloid ß (Aß) sequence, have dramatic impact on the risk for Alzheimer disease; A2V is causative, and A2T is protective. Assuming a crucial role of amyloid-Aß in neurodegeneration, we hypothesized that both A2V and A2T mutations cause distinct changes in Aß properties that may at least partially explain these completely different phenotypes. Using human APP-overexpressing primary neurons, we observed significantly decreased Aß production in the A2T mutant along with an enhanced Aß generation in the A2V mutant confirming earlier data from non-neuronal cell lines. More importantly, thioflavin T fluorescence assays revealed that the mutations, while having little effect on Aß42 peptide aggregation, dramatically change the properties of the Aß40 pool with A2V accelerating and A2T delaying aggregation of the Aß peptides. In line with the kinetic data, Aß A2T demonstrated an increase in the solubility at equilibrium, an effect that was also observed in all mixtures of the A2T mutant with the wild type Aß40. We propose that in addition to the reduced ß-secretase cleavage of APP, the impaired propensity to aggregate may be part of the protective effect conferred by A2T substitution. The interpretation of the protective effect of this mutation is thus much more complicated than proposed previously.

Biofilm-induced changes to the composite surface.

OBJECTIVES: Composites may undergo biodegradation in the oral cavity. The objective was to investigate the effect of single- and multi-species biofilms on the surface roughness and topography of two composites. METHODS: Disk-shaped specimens of a paste-like, Bis-GMA-free (Gradia Direct Anterior, GC), and a flowable, Bis-GMA-based composite (Tetric EvoFlow, Ivoclar-Vivadent) were prepared. After ethylene-oxide sterilization (38°C), specimens (n=3) were incubated with Streptococcus mutans or mixed bacterial culture (Streptococcus mutans, Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum). As negative controls, unexposed specimens and specimens exposed to sterile medium (BHI) were used. Specimens exposed to acidified BHI medium (pH=5) and enzymatic solution of cholesterol esterase served as positive control. Following 6-week incubation, the attached biofilms were collected for real-time PCR assessment, after which the surface roughness and topography of the specimens were analyzed with atomic force microscopy. Surface hydrophilicity/hydrophobicity was determined by contact angle measurements. Biofilm structure was analyzed with scanning electron microscopy. RESULTS: Even though multi-species biofilms were thicker, with more cells attached, they did not significantly affect the surface roughness of the composites. On the other hand, S. mutans alone significantly increased the roughness of Tetric by 40.3%, while its effect on Gradia was lower (12%). The total amount of attached bacteria, however, did not differ between the composites. CONCLUSIONS: S. mutans can increase the surface roughness of composites, depending on their composition. This ability of S. mutans is, however, mitigated in co-culture with other species. In particular, bacterial esterases seem to be responsible for the increased composite surface roughness upon biofilms exposure. CLINICAL SIGNIFICANCE: Cariogenic bacteria can degrade composites, thereby increasing the surface roughness. Increased roughness and subsequent improved bacterial accumulation may facilitate the development of secondary caries around composites, which is the most common reason for the restoration failure.

Amyloid beta oligomers induce neuronal elasticity changes in age-dependent manner: a force spectroscopy study on living hippocampal neurons.

Small soluble species of amyloid-beta (Aß) formed during early peptide aggregation stages are responsible for several neurotoxic mechanisms relevant to the pathology of Alzheimer's disease (AD), although their interaction with the neuronal membrane is not completely understood. This study quantifies the changes in the neuronal membrane elasticity induced by treatment with the two most common Aß isoforms found in AD brains: Aß40 and Aß42. Using quantitative atomic force microscopy (AFM), we measured for the first time the static elastic modulus of living primary hippocampal neurons treated with pre-aggregated Aß40 and Aß42 soluble species. Our AFM results demonstrate changes in the elasticity of young, mature and aged neurons treated for a short time with the two Aß species pre-aggregated for 2 hours. Neurons aging under stress conditions, showing aging hallmarks, are the most susceptible to amyloid binding and show the largest decrease in membrane stiffness upon Aß treatment. Membrane stiffness defines the way in which cells respond to mechanical forces in their environment and has been shown to be important for processes such as gene expression, ion-channel gating and neurotransmitter vesicle transport. Thus, one can expect that changes in neuronal membrane elasticity might directly induce functional changes related to neurodegeneration.

The Cancer Cell Oxygen Sensor PHD2 Promotes Metastasis via Activation of Cancer-Associated Fibroblasts.

Several questions about the role of the oxygen sensor prolyl-hydroxylase 2 (PHD2) in cancer have not been addressed. First, the role of PHD2 in metastasis has not been studied in a spontaneous tumor model. Here, we show that global PHD2 haplodeficiency reduced metastasis without affecting tumor growth. Second, it is unknown whether PHD2 regulates cancer by affecting cancer-associated fibroblasts (CAFs). We show that PHD2 haplodeficiency reduced metastasis via two mechanisms: (1) by decreasing CAF activation, matrix production, and contraction by CAFs, an effect that surprisingly relied on PHD2 deletion in cancer cells, but not in CAFs; and (2) by improving tumor vessel normalization. Third, the effect of concomitant PHD2 inhibition in malignant and stromal cells (mimicking PHD2 inhibitor treatment) is unknown. We show that global PHD2 haplodeficiency, induced not only before but also after tumor onset, impaired metastasis. These findings warrant investigation of PHD2's therapeutic potential.