A Comparative Study on the Lysosomal Cation Channel TMEM175 Using Automated Whole-Cell Patch-Clamp, Lysosomal Patch-Clamp, and Solid Supported Membrane-Based Electrophysiology: Functional Characterization and High-Throughput Screening Assay Development.

The lysosomal cation channel TMEM175 is a Parkinson's disease-related protein and a promising drug target. Unlike whole-cell automated patch-clamp (APC), lysosomal patch-clamp (LPC) facilitates physiological conditions, but is not yet suitable for high-throughput screening (HTS) applications. Here, we apply solid supported membrane-based electrophysiology (SSME), which enables both direct access to lysosomes and high-throughput electrophysiological recordings. In SSME, ion translocation mediated by TMEM175 is stimulated using a concentration gradient at a resting potential of 0 mV. The concentration-dependent K+ response exhibited an I/c curve with two distinct slopes, indicating the existence of two conducting states. We measured H+ fluxes with a permeability ratio of PH/PK = 48,500, which matches literature findings from patch-clamp studies, validating the SSME approach. Additionally, TMEM175 displayed a high pH dependence. Decreasing cytosolic pH inhibited both K+ and H+ conductivity of TMEM175. Conversely, lysosomal pH and pH gradients did not have major effects on TMEM175. Finally, we developed HTS assays for drug screening and evaluated tool compounds (4-AP, Zn as inhibitors; DCPIB, arachidonic acid, SC-79 as enhancers) using SSME and APC. Additionally, we recorded EC50 data for eight blinded TMEM175 enhancers and compared the results across all three assay technologies, including LPC, discussing their advantages and disadvantages.

Nickel and zinc micronutrient availability in Phanerozoic oceans.

Nickel and zinc are both bio-essential micronutrients with a nutrient-like distribution in the modern ocean, but show key differences in their biological functions and geochemical behavior. Eukaryotic phytoplankton, and especially diatoms, have high Zn quotas, whereas cyanobacteria generally require relatively more Ni. Secular changes in the relative availability of these micronutrients may, therefore, have affected the evolution and diversification of phytoplankton. In this study, we use a large compilation of Ni and Zn concentration data for Phanerozoic sediments to evaluate long-term changes in Ni and Zn availability and possible links to phytoplankton evolution. Modern data suggest that organic-rich sediments capture the dissolved deep ocean Ni/Zn ratio, regardless of local depositional conditions. We use this observation to constrain Ni/Zn ratios for past oceans, based on data from the sedimentary record. This record highlights long-term changes in the relative availability of these micronutrients that can be linked to the (bio)geochemical conditions on the Earth's surface. Early Palaeozoic oceans were likely relatively Ni rich, with sedimentary Ni/Zn ratios for this interval mostly being around ~1 or higher. A comparison with Phanerozoic strontium-, carbon-, and sulfur-isotopic records suggests that the late Palaeozoic decrease in sulfidic conditions and increase in hydrothermal inputs and organic-carbon burial rates caused a shift towards more Zn-rich conditions. Mesozoic and Cenozoic sediments show relatively Zn-rich oceans for these time intervals, with sedimentary Ni/Zn ratios mostly being around ~1 or lower. These observations imply that the diversification of the dominant groups of modern eukaryotic phytoplankton occurred in relatively Zn-rich oceans and that these organisms still carry this signature in their stoichiometries. However, the Phanerozoic transition to a more Zn-rich ocean pre-dates the origin and diversification of modern eukaryotes and, therefore, this transition was likely not the main direct cause for eukaryotic diversification in the Mesozoic and Cenozoic Eras.

Upper limits on the extent of seafloor anoxia during the PETM from uranium isotopes.

The Paleocene Eocene Thermal Maximum (PETM) represents a major carbon cycle and climate perturbation that was associated with ocean de-oxygenation, in a qualitatively similar manner to the more extensive Mesozoic Oceanic Anoxic Events. Although indicators of ocean de-oxygenation are common for the PETM, and linked to biotic turnover, the global extent and temporal progression of de-oxygenation is poorly constrained. Here we present carbonate associated uranium isotope data for the PETM. A lack of resolvable perturbation to the U-cycle during the event suggests a limited expansion of seafloor anoxia on a global scale. We use this result, in conjunction with a biogeochemical model, to set an upper limit on the extent of global seafloor de-oxygenation. The model suggests that the new U isotope data, whilst also being consistent with plausible carbon emission scenarios and observations of carbon cycle recovery, permit a maximum ~10-fold expansion of anoxia, covering <2% of seafloor area.

Brief oxygenation events in locally anoxic oceans during the Cambrian solves the animal breathing paradox.

Oxygen is a prerequisite for all large and motile animals. It is a puzzling paradox that fossils of benthic animals are often found in black shales with geochemical evidence for deposition in marine environments with anoxic and sulfidic bottom waters. It is debated whether the geochemical proxies are unreliable, affected by diagenesis, or whether the fossils are transported from afar or perhaps were not benthic. Here, we improved the stratigraphic resolution of marine anoxia records 100-1000 fold using core-scanning X-Ray Fluorescence and established a centennial resolution record of oxygen availability at the seafloor in an epicontinental sea that existed ~501-494 million years ago. The study reveals that anoxic bottom-water conditions, often with toxic hydrogen sulfide present, were interrupted by brief oxygenation events of 600-3000 years duration, corresponding to 1-5 mm stratigraphic thickness. Fossil shells occur in some of these oxygenated intervals suggesting that animals invaded when conditions permitted an aerobic life style at the seafloor. Although the fauna evidently comprised opportunistic species adapted to low oxygen environments, these findings reconcile a long-standing debate between paleontologists and geochemists, and shows the potential of ultra-high resolution analyses for reconstructing redox conditions in past oceans.

Perturbation to the nitrogen cycle during rapid Early Eocene global warming.

The degree to which ocean deoxygenation will alter the function of marine communities remains unclear but may be best constrained by detailed study of intervals of rapid warming in the geologic past. The Paleocene-Eocene Thermal Maximum (PETM) was an interval of rapid warming that was the result of increasing contents of greenhouse gases in the atmosphere that had wide ranging effects on ecosystems globally. Here, we present stable nitrogen isotope data from the Eastern Peri-Tethys Ocean that record a significant transition in the nitrogen cycle. At the initiation of the PETM, the nitrogen isotopic composition of sediments decreased by ~6‰ to as low as -3.4‰, signaling reorganization of the marine nitrogen cycle. Warming, changes in ocean circulation, and deoxygenation caused a transition to nitrogen cycle to conditions that were most similar to those experienced during Oceanic Anoxic Events of the Mesozoic.

Uranium isotope evidence for two episodes of deoxygenation during Oceanic Anoxic Event 2.

Oceanic Anoxic Event 2 (OAE 2), occurring ∼94 million years ago, was one of the most extreme carbon cycle and climatic perturbations of the Phanerozoic Eon. It was typified by a rapid rise in atmospheric CO2, global warming, and marine anoxia, leading to the widespread devastation of marine ecosystems. However, the precise timing and extent to which oceanic anoxic conditions expanded during OAE 2 remains unresolved. We present a record of global ocean redox changes during OAE 2 using a combined geochemical and carbon cycle modeling approach. We utilize a continuous, high-resolution record of uranium isotopes in pelagic and platform carbonate sediments to quantify the global extent of seafloor anoxia during OAE 2. This dataset is then compared with a dynamic model of the coupled global carbon, phosphorus, and uranium cycles to test hypotheses for OAE 2 initiation. This unique approach highlights an intra-OAE complexity that has previously been underconstrained, characterized by two expansions of anoxia separated by an episode of globally significant reoxygenation coincident with the "Plenus Cold Event." Each anoxic expansion event was likely driven by rapid atmospheric CO2 injections from multiphase Large Igneous Province activity.

Caffeine reverses cognitive impairment and decreases brain amyloid-beta levels in aged Alzheimer's disease mice.

We have recently shown that Alzheimer's disease (AD) transgenic mice given a moderate level of caffeine intake (the human equivalent of 5 cups of coffee per day) are protected from development of otherwise certain cognitive impairment and have decreased hippocampal amyloid-beta (Abeta) levels due to suppression of both beta-secretase (BACE1) and presenilin 1 (PS1)/gamma-secretase expression. To determine if caffeine intake can have beneficial effects in "aged" APPsw mice already demonstrating cognitive impairment, we administered caffeine in the drinking water of 18-19 month old APPsw mice that were impaired in working memory. At 4-5 weeks into caffeine treatment, those impaired transgenic mice given caffeine (Tg/Caff) exhibited vastly superior working memory compared to the continuing impairment of control transgenic mice. In addition, Tg/Caff mice had substantially reduced Abeta deposition in hippocampus (decrease 40%) and entorhinal cortex (decrease 46%), as well as correlated decreases in brain soluble Abeta levels. Mechanistically, evidence is provided that caffeine suppression of BACE1 involves the cRaf-1/NFkappaB pathway. We also determined that caffeine concentrations within human physiological range effectively reduce active and total glycogen synthase kinase 3 levels in SweAPP N2a cells. Even with pre-existing and substantial Abeta burden, aged APPsw mice exhibited memory restoration and reversal of AD pathology, suggesting a treatment potential of caffeine in cases of established AD.

Caffeine suppresses amyloid-beta levels in plasma and brain of Alzheimer's disease transgenic mice.

Recent epidemiologic studies suggest that caffeine may be protective against Alzheimer's disease (AD). Supportive of this premise, our previous studies have shown that moderate caffeine administration protects/restores cognitive function and suppresses brain amyloid-beta (Abeta) production in AD transgenic mice. In the present study, we report that acute caffeine administration to both young adult and aged AD transgenic mice rapidly reduces Abeta levels in both brain interstitial fluid and plasma without affecting Abeta elimination. Long-term oral caffeine treatment to aged AD mice provided not only sustained reductions in plasma Abeta, but also decreases in both soluble and deposited Abeta in hippocampus and cortex. Irrespective of caffeine treatment, plasma Abeta levels did not correlate with brain Abeta levels or with cognitive performance in individual aged AD mice. Although higher plasma caffeine levels were strongly associated with lower plasma Abeta1-40 levels in aged AD mice, plasma caffeine levels were also not linked to cognitive performance. Plasma caffeine and theophylline levels were tightly correlated, both being associated with reduced inflammatory cytokine levels in hippocampus. Our conclusion is two-fold: first, that both plasma and brain Abeta levels are reduced by acute or chronic caffeine administration in several AD transgenic lines and ages, indicating a therapeutic value of caffeine against AD; and second, that plasma Abeta levels are not an accurate index of brain Abeta levels/deposition or cognitive performance in aged AD mice.

Abeta-specific Th2 cells provide cognitive and pathological benefits to Alzheimer's mice without infiltrating the CNS.

We have found that a small number of purified Th2-biased Abeta-specific T cells are sufficient to provide profound cognitive and pathological benefits in an APP+PS1 mouse model for Alzheimer's disease. Six weeks after receiving T cell infusions, cognitively-impaired mice performed significantly better in working memory tasks, which correlated with higher plasma levels of soluble Abeta. Pathological analysis of the hippocampus revealed a 30% decrease of plaque-associated microglia and less vascular amyloidosis in T cell treated mice. The infusion of Abeta-specific Th2 cells also reduced plasma levels of IFN-gamma, TNF-alpha, GM-CSF, IL-2 and IL-4, which are elevated in untreated APP+PS1 mice. No significant immune cell infiltration and no anti-Abeta antibody titers occurred in the T cell treated mice. These results demonstrate that Abeta-specific Th2 cells are sufficient to reverse cognitive impairment and provide multiple pathological benefits in an Alzheimer's mouse model.