Role of the fast kinetics of pyroglutamate-modified amyloid-β oligomers in membrane binding and membrane permeability.

Membrane permeability to ions and small molecules is believed to be a critical step in the pathology of Alzheimer's disease (AD). Interactions of oligomers formed by amyloid-β (Aβ) peptides with the plasma cell membrane are believed to play a fundamental role in the processes leading to membrane permeability. Among the family of Aβs, pyroglutamate (pE)-modified Aβ peptides constitute the most abundant oligomeric species in the brains of AD patients. Although membrane permeability mechanisms have been studied for full-length Aβ1-40/42 peptides, these have not been sufficiently characterized for the more abundant AβpE3-42 fragment. Here we have compared the adsorbed and membrane-inserted oligomeric species of AβpE3-42 and Aβ1-42 peptides. We find lower concentrations and larger dimensions for both species of membrane-associated AβpE3-42 oligomers. The larger dimensions are attributed to the faster self-assembly kinetics of AβpE3-42, and the lower concentrations are attributed to weaker interactions with zwitterionic lipid headgroups. While adsorbed oligomers produced little or no significant membrane structural damage, increased membrane permeabilization to ionic species is understood in terms of enlarged membrane-inserted oligomers. Membrane-inserted AβpE3-42 oligomers were also found to modify the mechanical properties of the membrane. Taken together, our results suggest that membrane-inserted oligomers are the primary species responsible for membrane permeability

Genomic Dissection of Bipolar Disorder and Schizophrenia, Including 28 Subphenotypes

publisher: Elsevier articletitle: Genomic Dissection of Bipolar Disorder and Schizophrenia, Including 28 Subphenotypes journaltitle: Cell articlelink: https://doi.org/10.1016/j.cell.2018.05.046 content_type: article copyright: © 2018 Elsevier Inc

In pursuit of small molecule based prevention of Alzheimer's disease : defining the mechanistic pathway via bilayer electrophysiology

The prevailing hypothesis for pathology of Alzheimer's disease (AD) proposes that amyloid-beta (A[Beta]) peptides are the toxic element. Small oligomers of A[Beta] are believed to induce uncontrolled, neurotoxic ion flux across cellular membranes. The resulting inability of neurons to regulate their intracellular concentration of ions, particularly calcium ions, has been associated with cell death and may contribute to the cognitive impairment typically seen in AD patients. The mechanism of the ion flux is not fully understood, but the most direct mechanism of membrane disruption would be the formation of channel-like pores. Structural models and experimental evidence suggest that A[Beta] pores form from an assembly of loosely-associated mobile [Beta]-sheet subunits. However, further understanding of the underlying mechanism of AD will be crucial to our ability to design highly efficacious therapeutics. The dissertation focuses on the application of bilayer electrophysiological recordings to the study and determination of the mechanistic pathway of A[Beta] pathophysiology in AD. Biophysical characterization of the highly cytotoxic pyroglutamate- modified A[Beta] (A[Beta]pE) is first presented. We show that A[Beta]pE activity is shifted to higher conductance events, which could explain their increased toxicity. Next, we introduce, and provide proof of concept for, a newly developed analysis tool which more accurately depicts cytotoxicity than traditional channel analysis methods. Finally, we use two small molecule drug candidates to test efficacy and gain insight into the mechanism of pore formation. From the results we suggest structural targets for future therapeutic design. This work advances the understanding of the underlying mechanism of AD pathophysiology by pore formation and will aid the design of small molecule pharmaceuticals for the treatment/ prevention of this devastating diseas

Graphene nanopore support system for simultaneous high-resolution AFM imaging and conductance measurements.

Accurately defining the nanoporous structure and sensing the ionic flow across nanoscale pores in thin films and membranes has a wide range of applications, including characterization of biological ion channels and receptors, DNA sequencing, molecule separation by nanoparticle films, sensing by block co-polymers films, and catalysis through metal-organic frameworks. Ionic conductance through nanopores is often regulated by their 3D structures, a relationship that can be accurately determined only by their simultaneous measurements. However, defining their structure-function relationships directly by any existing techniques is still not possible. Atomic force microscopy (AFM) can image the structures of these pores at high resolution in an aqueous environment, and electrophysiological techniques can measure ion flow through individual nanoscale pores. Combining these techniques is limited by the lack of nanoscale interfaces. We have designed a graphene-based single-nanopore support (∼5 nm thick with ∼20 nm pore diameter) and have integrated AFM imaging and ionic conductance recording using our newly designed double-chamber recording system to study an overlaid thin film. The functionality of this integrated system is demonstrated by electrical recording (<10 pS conductance) of suspended lipid bilayers spanning a nanopore and simultaneous AFM imaging of the bilayer

Computational Methods for Structural and Functional Studies of Alzheimer’s Amyloid Ion Channels

Aggregation can be studied by a range of methods, experimental and computational. Aggregates form in solution, across solid surfaces, and on and in the membrane, where they may assemble into unregulated leaking ion channels. Experimental probes of ion channel conformations and dynamics are challenging. Atomistic molecular dynamics (MD) simulations are capable of providing insight into structural details of amyloid ion channels in the membrane at a resolution not achievable experimentally. Since data suggest that late stage Alzheimer's disease involves formation of toxic ion channels, MD simulations have been used aiming to gain insight into the channel shapes, morphologies, pore dimensions, conformational heterogeneity, and activity. These can be exploited for drug discovery. Here we describe computational methods to model amyloid ion channels containing the β-sheet motif at atomic scale and to calculate toxic pore activity in the membrane

Computational Methods for Structural and Functional Studies of Alzheimer's Amyloid Ion Channels.

Aggregation can be studied by a range of methods, experimental and computational. Aggregates form in solution, across solid surfaces, and on and in the membrane, where they may assemble into unregulated leaking ion channels. Experimental probes of ion channel conformations and dynamics are challenging. Atomistic molecular dynamics (MD) simulations are capable of providing insight into structural details of amyloid ion channels in the membrane at a resolution not achievable experimentally. Since data suggest that late stage Alzheimer's disease involves formation of toxic ion channels, MD simulations have been used aiming to gain insight into the channel shapes, morphologies, pore dimensions, conformational heterogeneity, and activity. These can be exploited for drug discovery. Here we describe computational methods to model amyloid ion channels containing the β-sheet motif at atomic scale and to calculate toxic pore activity in the membrane

Small molecule NPT-440-1 inhibits ionic flux through Aβ1-42 pores: Implications for Alzheimer's disease therapeutics.

Increased levels of soluble amyloid-beta (Aβ) oligomers are suspected to underlie Alzheimer's disease (AD) pathophysiology. These oligomers have been shown to form multi-subunit Aβ pores in bilayers and induce uncontrolled, neurotoxic, ion flux, particularly calcium ions, across cellular membranes that might underlie cognitive impairment in AD. Small molecule interventions that modulate pore activity could effectively prevent or ameliorate their toxic activity. Here we examined the efficacy of a small molecule, NPT-440-1, on modulating amyloid pore permeability. Co-incubation of B103 rat neuronal cells with NPT-440-1 and Aβ1-42 prevented calcium influx. In purified lipid bilayers, we show that a 10-15min preincubation, prior to membrane introduction, was required to prevent conductance. Thioflavin-T and circular dichroism both suggested a reduction in Aβ1-42 β-sheet content during this incubation period. Combined with previous studies on site-specific amino acid substitutions, these results suggest that pharmacological modulation of Aβ1-42 could prevent amyloid pore-mediated AD pathogenesis

Small molecule NPT-440-1 inhibits ionic flux through Aβ1-42 pores: Implications for Alzheimer's disease therapeutics.

Increased levels of soluble amyloid-beta (Aβ) oligomers are suspected to underlie Alzheimer's disease (AD) pathophysiology. These oligomers have been shown to form multi-subunit Aβ pores in bilayers and induce uncontrolled, neurotoxic, ion flux, particularly calcium ions, across cellular membranes that might underlie cognitive impairment in AD. Small molecule interventions that modulate pore activity could effectively prevent or ameliorate their toxic activity. Here we examined the efficacy of a small molecule, NPT-440-1, on modulating amyloid pore permeability. Co-incubation of B103 rat neuronal cells with NPT-440-1 and Aβ1-42 prevented calcium influx. In purified lipid bilayers, we show that a 10-15min preincubation, prior to membrane introduction, was required to prevent conductance. Thioflavin-T and circular dichroism both suggested a reduction in Aβ1-42 β-sheet content during this incubation period. Combined with previous studies on site-specific amino acid substitutions, these results suggest that pharmacological modulation of Aβ1-42 could prevent amyloid pore-mediated AD pathogenesis