Daphnetin ameliorates Aß pathogenesis via STAT3/GFAP signaling in an APP/PS1 double-transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) has become a major public health problem that affects the elderly population. Therapeutic compounds with curative effects are not available due to the complex pathogenesis of AD. Daphnetin, a natural coumarin derivative and inhibitor of various kinases, has anti-inflammatory and antioxidant activities. In this study, we found that daphnetin improved spatial learning and memory in an amyloid precursor protein (APP)/presenilin 1 (PS1) double-transgenic mouse model of AD. Daphnetin markedly decreased the levels of amyloid-ß peptide 1-40 (Aß40) and 1-42 (Aß42) in the cerebral cortex, downregulated the expressions of enzymes involved in APP processing, e.g., beta-site APP-cleaving enzyme (BACE), nicastrin and presenilin enhancer protein 2 (PEN2). We further found the reduced serum levels of inflammatory factors, including interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and chemokine (C-C motif) ligand 3 (CCL3), while daphnetin increased total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) levels in the serum. Interestingly, daphnetin markedly decreased the expression of glial fibrillary acidic protein (GFAP) and the upstream regulatory molecule- phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in APP/PS1 mice, and mainly inhibited the phosphorylation of STAT3 at Ser727 to decrease GFAP expression evidenced in a LPS-activated glial cell model. These results suggest that daphnetin ameliorates cognitive deficits and that Aß deposition in APP/PS1 mice is mainly correlated with astrocyte activation and APP processing.

Safflower leaf ameliorates cognitive impairment through moderating excessive astrocyte activation in APP/PS1 mice.

In addition to beta-amyloid (Aß) plaques and neurofibrillary tangles, Alzheimer's disease (AD) is typically triggered or accompanied by abnormal inflammation, oxidative stress and astrocyte activation. Safflower (Carthamus tinctorius L.) leaf, featuring functional ingredients, is a commonly consumed leafy vegetable. Whether and how dietary safflower leaf powder (SLP) ameliorates cognitive function in an AD mouse model has remained minimally explored. Therefore, we orally administered SLP to APP/PS1 transgenic mice to explore the neuroprotective effects of SLP in preventing AD progression. We found that SLP markedly improved cognitive impairment in APP/PS1 mice, as indicated by the water maze test. We further demonstrated that SLP treatment ameliorated inflammation, oxidative stress and excessive astrocyte activation. Further investigation indicated that SLP decreased the Aß burden in APP/PS1 mice by mediating excessive astrocyte activation. Our study suggests that safflower leaf is possibly a promising, cognitively beneficial food for preventing and alleviating AD-related dementia.

Topological reorganizations of mitochondria isolated from rat brain after 72 hours of paradoxical sleep deprivation, revealed by electron cryo-tomography.

Sleep deprivation has profound influence on several aspects of health and disease. Mitochondria dysfunction has been implicated to play an essential role in the neuronal cellular damage induced by sleep deprivation, but little is known about how neuronal mitochondrial ultrastructure is affected under sleep deprivation. In this report, we utilized electron cryo-tomography to reconstruct the three-dimensional (3-D) mitochondrial structure and extracted morphometric parameters to quantitatively characterize its reorganizations. Isolated mitochondria from the hippocampus and cerebral cortex of adult male Sprague-Dawley rats after 72 h of paradoxical sleep deprivation (PSD) were reconstructed and analyzed. Statistical analysis of six morphometric parameters specific to the mitochondrial inner membrane topology revealed identical pattern of changes in both the hippocampus and cerebral cortex but with higher significance levels in the hippocampus. The structural differences were indistinguishable by conventional phenotypic methods based on two-dimensional electron microscopy images or 3-D electron tomography reconstructions. Furthermore, to correlate structure alterations with mitochondrial functions, high-resolution respirometry was employed to investigate the effects of PSD on mitochondrial respiration, which showed that PSD significantly suppressed the mitochondrial respiratory capacity of the hippocampus, whereas the isolated mitochondria from the cerebral cortex were less affected. These results demonstrate the capability of the morphometric parameters for quantifying complex structural reorganizations and suggest a correlation between PSD and inner membrane architecture/respiratory functions of the brain mitochondria with variable effects in different brain regions.

Single-Molecule Fluorescence Measurement of SNARE-Mediated Vesicle Fusion.

This chapter expounds the single vesicle-vesicle fluorescence resonance energy transfer (FRET) measurement to study the membrane fusion mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. The formation of a four-α-helix bundle of SNARE proteins can drive two membranes to a close proximity for fusion. Through single-molecule FRET-based microscopy, the lipid-mixing process at the single-vesicle level can be tracked in real time. This reconstitution system is applicable to study the molecular mechanism of SNAREs during different membrane fusion stages, such as docking, hemifusion, and full fusion. Four main parts are described in this chapter, including SNARE reconstitution, imaging preparation, data collection, and analysis.

N-Terminal Acetylation Preserves α-Synuclein from Oligomerization by Blocking Intermolecular Hydrogen Bonds.

The abnormal aggregation of α-synuclein (α-Syn) is closely associated with Parkinson's disease. Different post-translational modifications of α-Syn have been identified and contribute distinctly in α-Syn aggregation and cytotoxicity. Recently, α-Syn was reported to be N-terminally acetylated in cells, yet the functional implication of this modification, especially in α-Syn oligomerization, remains unclear. By using a solid-state nanopore system, we found that N-terminal acetylation can significantly decrease α-Syn oligomerization. Replica-exchange molecular dynamics simulations further revealed that addition of an acetyl group at the N-terminus disrupts intermolecular hydrogen bonds, which slows down the initial α-Syn oligomerization. Our finding highlights the essential role of N-terminal acetylation of α-Syn in preserving its native conformation against pathological aggregation.

SNARE-Reconstituted Liposomes as Controllable Zeptoliter Nanoreactors for Macromolecules.

Liposomes are synthetic phospholipid vesicles containing an aqueous lumen confined by a lipid bilayer. These vesicles have been used as nanoreactors because they offer confined environments that can result in significant changes to chemistry. However, a major limitation of using liposomes as nanocontainers is the impermeability of their lipid membranes. To overcome this, scientists have tested the use of porous liposomes generated by membrane phase changes or by pore formation proteins. In this study, the selective permeability of porous liposomes to molecules smaller than the threshold pore size is demonstrated for triggering internal reactions. Furthermore, in order to deliver macromolecules larger than the threshold pore size into lipid-based nanoreactors, fusogenic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins were employed to mediate membrane fusion between two liposomes to initiate reactions on demand without losing previously encapsulated macromolecules. This novel approach circumvents limitations associated with inserting molecules larger than the threshold pore size of porous lipid membranes.

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes.

In presynaptic nerve terminals, complexin regulates spontaneous "mini" neurotransmitter release and activates Ca2+-triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using single-particle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca2+-independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents.