Localized electrical stimulation triggers cell-type-specific proliferation in biofilms.

Biological systems ranging from bacteria to mammals utilize electrochemical signaling. Although artificial electrochemical signals have been utilized to characterize neural tissue responses, the effects of such stimuli on non-neural systems remain unclear. To pursue this question, we developed an experimental platform that combines a microfluidic chip with a multielectrode array (MiCMA) to enable localized electrochemical stimulation of bacterial biofilms. The device also allows for the simultaneous measurement of the physiological response within the biofilm with single-cell resolution. We find that the stimulation of an electrode locally changes the ratio of the two major cell types comprising Bacillus subtilis biofilms, namely motile and extracellular-matrix-producing cells. Specifically, stimulation promotes the proliferation of motile cells but not matrix cells, even though these two cell types are genetically identical and reside in the same microenvironment. Our work thus reveals that an electronic interface can selectively target bacterial cell types, enabling the control of biofilm composition and development.

The diphenylpyrazole compound anle138b blocks Aß channels and rescues disease phenotypes in a mouse model for amyloid pathology.

Alzheimer's disease is a devastating neurodegenerative disease eventually leading to dementia. An effective treatment does not yet exist. Here we show that oral application of the compound anle138b restores hippocampal synaptic and transcriptional plasticity as well as spatial memory in a mouse model for Alzheimer's disease, when given orally before or after the onset of pathology. At the mechanistic level, we provide evidence that anle138b blocks the activity of conducting Aß pores without changing the membrane embedded Aß-oligomer structure. In conclusion, our data suggest that anle138b is a novel and promising compound to treat AD-related pathology that should be investigated further.

Amyloid ß Ion Channels in a Membrane Comprising Brain Total Lipid Extracts.

Amyloid ß (Aß) oligomers are the predominant toxic species in the pathology of Alzheimer's disease. The prevailing mechanism for toxicity by Aß oligomers includes ionic homeostasis destabilization in neuronal cells by forming ion channels. These channel structures have been previously studied in model lipid bilayers. In order to gain further insight into the interaction of Aß oligomers with natural membrane compositions, we have examined the structures and conductivities of Aß oligomers in a membrane composed of brain total lipid extract (BTLE). We utilized two complementary techniques: atomic force microscopy (AFM) and black lipid membrane (BLM) electrical recording. Our results indicate that Aß1-42 forms ion channel structures in BTLE membranes, accompanied by a heterogeneous population of ionic current fluctuations. Notably, the observed current events generated by Aß1-42 peptides in BTLE membranes possess different characteristics compared to current events generated by the presence of Aß1-42 in model membranes comprising a 1:1 mixture of DOPS and POPE lipids. Oligomers of the truncated Aß fragment Aß17-42 (p3) exhibited similar ion conductivity behavior as Aß1-42 in BTLE membranes. However, the observed macroscopic ion flux across the BTLE membranes induced by Aß1-42 pores was larger than for p3 pores. Our analysis of structure and conductance of oligomeric Aß pores in a natural lipid membrane closely mimics the in vivo cellular environment suggesting that Aß pores could potentially accelerate the loss of ionic homeostasis and cellular abnormalities. Hence, these pore structures may serve as a target for drug development and therapeutic strategies for AD treatment.

Small molecule NPT-440-1 inhibits ionic flux through Aß1-42 pores: Implications for Alzheimer's disease therapeutics.

Increased levels of soluble amyloid-beta (Aß) oligomers are suspected to underlie Alzheimer's disease (AD) pathophysiology. These oligomers have been shown to form multi-subunit Aß 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 Aß1-42 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 Aß1-42 ß-sheet content during this incubation period. Combined with previous studies on site-specific amino acid substitutions, these results suggest that pharmacological modulation of Aß1-42 could prevent amyloid pore-mediated AD pathogenesis.

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 ß-sheet motif at atomic scale and to calculate toxic pore activity in the membrane.

Activity and architecture of pyroglutamate-modified amyloid-ß (AßpE3-42) pores.

Among the family of Aß peptides, pyroglutamate-modified Aß (AßpE) peptides are particularly associated with cytotoxicity in Alzheimer's disease (AD). They represent the dominant fraction of Aß oligomers in the brains of AD patients, but their accumulation in the brains of elderly individuals with normal cognition is significantly lower. Accumulation of AßpE plaques precedes the formation of plaques of full-length Aß (Aß1-40/42). Most of these properties appear to be associated with the higher hydrophobicity of AßpE as well as an increased resistance to enzymatic degradation. However, the important question of whether AßpE peptides induce pore activity in lipid membranes and their potential toxicity compared with other Aß pores is still open. Here we examine the activity of AßpE pores in anionic membranes using planar bilayer electrical recording and provide their structures using molecular dynamics simulations. We find that AßpE pores spontaneously induce ionic current across the membrane and have some similar properties to the other previously studied pores of the Aß family. However, there are also some significant differences. The onset of AßpE3-42 pore activity is generally delayed compared with Aß1-42 pores. However, once formed, AßpE3-42 pores produce increased ion permeability of the membrane, as indicated by a greater occurrence of higher conductance electrical events. Structurally, the lactam ring of AßpE peptides induces a change in the conformation of the N-terminal strands of the AßpE3-42 pores. While the N-termini of wild-type Aß1-42 peptides normally reside in the bulk water region, the N-termini of AßpE3-42 peptides tend to reside in the hydrophobic lipid core. These studies provide a first step to an understanding of the enhanced toxicity attributed to AßpE peptides.

Role of the fast kinetics of pyroglutamate-modified amyloid-ß 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-ß (Aß) peptides with the plasma cell membrane are believed to play a fundamental role in the processes leading to membrane permeability. Among the family of Aßs, pyroglutamate (pE)-modified Aß peptides constitute the most abundant oligomeric species in the brains of AD patients. Although membrane permeability mechanisms have been studied for full-length Aß1-40/42 peptides, these have not been sufficiently characterized for the more abundant AßpE3-42 fragment. Here we have compared the adsorbed and membrane-inserted oligomeric species of AßpE3-42 and Aß1-42 peptides. We find lower concentrations and larger dimensions for both species of membrane-associated AßpE3-42 oligomers. The larger dimensions are attributed to the faster self-assembly kinetics of AßpE3-42, 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 AßpE3-42 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.

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.