Reciprocal modulation between amyloid precursor protein and synaptic membrane cholesterol revealed by live cell imaging.

The amyloid precursor protein (APP) has been extensively studied because of its association with Alzheimer's disease (AD). However, APP distribution across different subcellular membrane compartments and its function in neurons remains unclear. We generated an APP fusion protein with a pH-sensitive green fluorescent protein at its ectodomain and a pH-insensitive blue fluorescent protein at its cytosolic domain and used it to measure APP's distribution, subcellular trafficking, and cleavage in live neurons. This reporter, closely resembling endogenous APP, revealed only a limited correlation between synaptic activities and APP trafficking. However, the synaptic surface fraction of APP was increased by a reduction in membrane cholesterol levels, a phenomenon that involves APP's cholesterol-binding motif. Mutations at or near binding sites not only reduced both the surface fraction of APP and membrane cholesterol levels in a dominant negative manner, but also increased synaptic vulnerability to moderate membrane cholesterol reduction. Our results reveal reciprocal modulation of APP and membrane cholesterol levels at synaptic boutons.

Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters.

Higher-order structures of the microtubule (MT) cytoskeleton are comprised of two architectures: bundles and asters. Although both architectures are critical for cellular function, the molecular pathways that drive aster formation are poorly understood. Here, we study aster formation by human minus-end-directed kinesin-14 (HSET/KIFC1). We show that HSET is incapable of forming asters from preformed, nongrowing MTs, but rapidly forms MT asters in the presence of soluble (non-MT) tubulin. HSET binds soluble (non-MT) tubulin via its N-terminal tail domain to form heterogeneous HSET-tubulin clusters containing multiple motors. Cluster formation induces motor processivity and rescues the formation of asters from nongrowing MTs. We then show that excess soluble (non-MT) tubulin stimulates aster formation in HeLa cells overexpressing HSET during mitosis. We propose a model where HSET can toggle between MT bundle and aster formation in a manner governed by the availability of soluble (non-MT) tubulin.

Ammonium chloride alters neuronal excitability and synaptic vesicle release.

Genetically encoded pH-sensors are widely used in studying cell membrane trafficking and membrane protein turnover because they render exo-/endocytosis-associated pH changes to fluorescent signals. For imaging and analysis purposes, high concentration ammonium chloride is routinely used to alkalize intracellular membrane compartments under the assumption that it does not cause long-term effects on cellular processes being studied like neurotransmission. However, pathological studies about hyperammonemia have shown that ammonium is toxic to brain cells especially astrocytes and neurons. Here, we focus on ammonium's physiological impacts on neurons including membrane potential, cytosolic Ca2+ and synaptic vesicles. We have found that extracellularly applied ammonium chloride as low as 5 mM causes intracellular Ca2+-increase and a reduction of vesicle release even after washout. The often-used 50 mM ammonium chloride causes more extensive and persistent changes, including membrane depolarization, prolonged elevation of intracellular Ca2+ and diminution of releasable synaptic vesicles. Our findings not only help to bridge the discrepancies in previous studies about synaptic vesicle release using those pH-sensors or other vesicle specific reporters, but also suggest an intriguing relationship between intracellular pH and neurotransmission.