Shifting the balance: soluble ADAM10 as a potential treatment for Alzheimer's disease.

Introduction: Accumulation of amyloid ß in the brain is regarded as a key initiator of Alzheimer's disease pathology. Processing of the amyloid precursor protein (APP) in the amyloidogenic pathway yields neurotoxic amyloid ß species. In the non-amyloidogenic pathway, APP is processed by membrane-bound ADAM10, the main α-secretase in the nervous system. Here we present a new enzymatic approach for the potential treatment of Alzheimer's disease using a soluble form of ADAM10. Methods: The ability of the soluble ADAM10 to shed overexpressed and endogenous APP was determined with an ADAM10 knockout cell line and a human neuroblastoma cell line, respectively. We further examined its effect on amyloid ß aggregation by thioflavin T fluorescence, HPLC, and confocal microscopy. Using N-terminal and C-terminal enrichment proteomic approaches, we identified soluble ADAM10 substrates. Finally, a truncated soluble ADAM10, based on the catalytic domain, was expressed in Escherichia coli for the first time, and its activity was evaluated. Results: The soluble enzyme hydrolyzes APP and releases the neuroprotective soluble APPα when exogenously added to cell cultures. The soluble ADAM10 inhibits the formation and aggregation of characteristic amyloid ß extracellular neuronal aggregates. The proteomic investigation identified new and verified known substrates, such as VGF and N-cadherin, respectively. The truncated variant also exhibited α-secretase capacity as shown with a specific ADAM10 fluorescent substrate in addition to shedding overexpressed and endogenous APP. Discussion: Our in vitro study demonstrates that exogenous treatment with a soluble variant of ADAM10 would shift the balance toward the non-amyloidogenic pathway, thus utilizing its natural neuroprotective effect and inhibiting the main neurotoxic amyloid ß species. The potential of such a treatment for Alzheimer's disease needs to be further evaluated in vivo.

Sub-inhibitory membrane damage undermines Staphylococcus aureus virulence.

We investigated antibacterial properties of a recently described membrane-active lipopeptide, C10OOc12O (decanoyl-ornithyl-ornithyl-dodecanoyl-ornithyl-amide) against Gram-positive bacteria (GPB). Minimal inhibitory concentrations (MICs) and kinetics were compared in culture media and plasma. Chemo-sensitization to antibiotics was determined using the checkerboard assay. Membrane damages were estimated using diverse membrane potential sensitive dyes. ATP levels and relevant enzymes activities were measured using commercial bioassay kits. While relatively weakly active in simple culture media, sub-MIC levels (~ten-fold) of C10OOc12O have significantly improved the antibacterial function of Human plasma. Mechanistic studies indicated that C10OOc12O-treated bacteria have sustained mild membrane damage(s) in association with rapid (within 2 min) but low (<10%) dissipation of the trans-membrane potential; Intracellular ATP levels were transiently reduced (~20%) whereas extracellular ATP increased only at MIC values; Sub-inhibitory concentrations were sufficient for inhibiting major agr-regulated virulence factors (lipase and α-toxin) and for sensitizing MRSA USA300 to the antibiotic oxacillin to the point of reverting the bacteria status from oxacillin-resistant to oxacillin-sensitive (i.e., oxacillin MIC was reduced from 32 to 0.1 mg/l). These findings argue that by means of mild depolarization, C10OOc12O affects the quorum sensing regulator in a manner that transiently weakens bacterial defenses, thereby enforcing studies that support the potential usefulness of fighting S. aureus (and possibly other GPB) infections, by targeting its virulence.