Serotonin, an importants ignalingm olecule in humans, has an unexpectedly high lipid membrane affinity. The significance of this finding has evokedc onsiderable speculation. Here we show that membrane binding by serotonin can directly modulate membrane properties and cellular function, providing an activity pathway completelyi ndependent of serotonin receptors. Atomic force microscopy shows that serotonin makes artificial lipid bilayers softer,a nd inducesn ucleation of liquid disordered domains inside the raft-likel iquid-ordered domains. Solid-state NMR spectrosco-py corroborates this data at the atomic level,r evealing ah omogeneous decrease in the order parameter of the lipid chainsi nt he presence of serotonin. In the RN46A immortalized serotonergicn euronal cell line, extracellular serotonin enhances transferrin receptor endocytosis, even in the presence of broad-spectrum serotonin receptor and transporter inhibitors. Similarly,i ti ncreases the membrane binding and internalizationo fo ligomeric peptides. Our resultsu ncover a mode of serotonin-membrane interaction that can potentiate key cellular processes in ar eceptor-independent fashion.

While the roles of intrinsically disordered protein domains in driving many interactions are increasingly well‐appreciated, the mechanism of toxicity of disease‐causing disordered proteins remains poorly understood. A prime example is Alzheimer's disease (AD) associated amyloid beta (Aβ). Aβ oligomers are highly toxic partially structured peptide assemblies with a distinct ordered region (residues ~10–40) and a shorter disordered region (residues ~1–9). Here, we investigate the role of this disordered domain and its relation to the ordered domain in the manifestation of toxicity through a set of Aβ fragments and stereo‐isomers designed for this purpose. We have measured their effects on lipid membranes and cultured neurons, probing their toxicity, intracellular distributions, and specific molecular interactions using the techniques of confocal imaging, lattice light sheet imaging, fluorescence lifetime imaging, and fluorescence correlation spectroscopy (FCS). Remarkably, we find that neither part ‐ Aβ10–40 or Aβ1–9, is toxic by itself. The ordered part (Aβ10–40) is the major determinant of how Aβ attaches to lipid bilayers, enters neuronal cells, and localizes primarily in the late endosomal compartments. However, once Aβ enters the cell, it is the disordered part (only when it is connected to the rest of the peptide) which has a strong and stereospecific interaction with an unknown cellular component, as demonstrated by distinct changes in the fluorescence lifetime of a fluorophore attached to the N‐terminal. This interaction appears to commit Aβ to the toxic pathway. Our findings correlate well with Aβ sites of familial AD mutations, a significant fraction of which cluster in the disordered region. We conclude that while the ordered region dictates attachment and cellular entry, the key to toxicity lies in the ordered part presenting the disordered part for a specific cellular interaction. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

No abstract

A␤ self-assembles into parallel cross-␤ fibrillar aggregates, which is associated with Alzheimer's disease pathology. A central hairpin turn around residues 23-29 is a defining characteristic of A␤ in its aggregated state. Major biophysical properties of A␤, including this turn, remain unaltered in the central fragment A␤ 18 -35 . Here, we synthesize a single deletion mutant, ⌬G25, with the aim of sterically hindering the hairpin turn in A␤ 18 -35 . We find that the solubility of the peptide goes up by more than 20-fold. Although some oligomeric structures do form, solution state NMR spectroscopy shows that they have mostly random coil conformations. Fibrils ultimately form at a much higher concentration but have widths approximately twice that of A␤ 18 -35 , suggesting an opening of the hairpin bend. Surprisingly, two-dimensional solid state NMR shows that the contact between Phe 19 and Leu 34 residues, observed in full-length A␤ and A␤ 18 -35 , is still intact in these fibrils. This is possible if the monomers in the fibril are arranged in an antiparallel ␤-sheet conformation. Indeed, IR measurements, supported by tyrosine cross-linking experiments, provide a characteristic signature of the antiparallel ␤-sheet. We conclude that the self-assembly of A␤ is critically dependent on the hairpin turn and on the contact between the Phe 19 and Leu 34 regions, making them potentially sensitive targets for Alzheimer's therapeutics. Our results show the importance of specific conformations in an aggregation process thought to be primarily driven by nonspecific hydrophobic interactions.Alzheimer's disease pathology has been associated with the aggregation of amyloid ␤ (A␤), 6 a 39 -43-amino acid-long peptide (1, 2). In this process, the unstructured monomers of A␤ get converted into amyloid fibrils composed of hairpin-shaped monomeric units assembled in a parallel ␤ sheet arrangement (3, 4). The central part of this hairpin structure consists of a hydrophilic region flanked by hydrophobic ␤-sheet-forming segments at both ends (5-10). This particular shape is believed to be dictated by the specific pattern of hydrophobic and charged residues in the A␤ sequence and has been identified even in soluble A␤ aggregates (7,(11)(12)(13)(14)(15)(16)(17), including the very early stage oligomers of A␤ (18 , and stabilizes the soluble monomeric and oligomeric assemblies of A␤ (19). This hairpin turn thus seems to be a critical factor dictating the self-assembly of A␤ under physiological conditions. Studying the influence of the turn region on the properties of A␤, separately from that of the distal terminal regions and without changing the electrostatics, can yield valuable information on the logic of A␤ assembly.A␤ aggregation appears to be primarily hydrophobic in nature. In fact a contact between hydrophobic regions containing Phe 19 and Leu 34 is one of the earliest contacts formed during the aggregation of amyloid ␤ (18). In a generic hydrophobic aggregate, the burial of the hydrophobic surface can in principle proceed in an ...

Designer receptors exclusively activated by designer drugs (DREADD)-based chemogenetic tools are extensively used to manipulate neuronal activity in a cell type-specific manner. Whole-cell patch-clamp recordings indicate membrane depolarization, coupled with increased neuronal firing rate, following administration of the DREADD ligand, clozapine-N-oxide (CNO) to activate the Gq-coupled DREADD, hM3Dq. Although hM3Dq has been used to enhance neuronal firing in order to manipulate diverse behaviors, often within 30 min to 1 h after CNO administration, the physiological effects on excitatory neurotransmission remain poorly understood. We investigated the influence of CNO-mediated hM3Dq DREADD activation on distinct aspects of hippocampal excitatory neurotransmission at the Schaffer collateral-CA1 synapse in hippocampal slices derived from mice expressing hM3Dq in Ca2+/calmodulin-dependent protein kinase α (CamKIIα)-positive excitatory neurons. Our results indicate a clear dose-dependent effect on field EPSP (fEPSP) slope, with no change noted at the lower dose of CNO (1 μM) and a significant, long-term decline in fEPSP slope observed at higher doses (5–20 μM). Further, we noted a robust θ burst stimulus (TBS) induced long-term potentiation (LTP) in the presence of the lower CNO (1 μM) dose, which was significantly attenuated at the higher CNO (20 μM) dose. Whole-cell patch-clamp recording revealed both complex dose-dependent regulation of excitability, and spontaneous and evoked activity of CA1 pyramidal neurons in response to hM3Dq activation across CNO concentrations. Our data indicate that CNO-mediated activation of the hM3Dq DREADD results in dose-dependent regulation of excitatory hippocampal neurotransmission and highlight the importance of careful interpretation of behavioral experiments involving chemogenetic manipulation.