Biology subjects

Demuro, A.

Publications and source records attributed to Demuro, A..

6 recordsLinked to original sources

bioRxiv2022

Modeling the kinetics of amyloid beta pores and long-term evolution of their Ca2+ toxicity

The formation of cation-permeable pores due to amyloid beta (A{beta}) in the plasma membrane (PM) and membranes of intracellular organelles has been shown to cause disruption of Ca2+ homeostasis in Alzheimers disease, leading to neuronal malfunction and degeneration. However, detailed information about the formation, function, and time-evolution of A{beta} pores are still lacking. Here we present a comprehensive analysis to show that the Ca2+ toxicity of PM pores formed by A{beta}42 progressively increases over the duration of the experiments that last for more than an hour. This mainly results from a progressively increasing number of pores and activity of individual pores having higher open probability (Po) and Ca2+ conductance. To gain deeper insight into the function and evolution of A{beta}42 pores and establish their role in Ca2+ toxicity beyond experimental times scales, we developed a data-driven computational model that not only mimics the kinetics of these pores at a given time but also tracks their time-evolution over many hours. Our model suggests that the rising toxicity of A{beta}42 pores over time is a combined result of the large number of short-lived pores with low Ca2+ conductance and long-lived higher-conductance pores but fewer in number. Analysis and modeling of the voltage-dependent data show that A{beta}42 pores are most active when the cells membrane potential is near resting values, making the cell at rest especially more prone to Ca2+ disruptions.

cell biology↗

bioRxiv2022

Intracellular injection of brain extracts from Alzheimer's disease patients trigger unregulated Ca2+ release from intracellular stores that hinders cellular bioenergetics

Strong evidence indicates that amyloid beta (A{beta}) inflicts its toxicity in Alzheimers disease (AD) by promoting uncontrolled elevation of cytosolic Ca2+ in neurons. We have previously shown that synthetic A{beta}42 oligomers stimulate abnormal intracellular Ca2+ release from the endoplasmic reticulum stores, suggesting that a similar mechanism of Ca2+ toxicity may be common to the endogenous A{beta}s oligomers. To investigate this possibility, we use human postmortem brain extracts from control and AD-affected patients and test their ability to trigger Ca2+ fluxes when injected intracellularly into Xenopus oocytes. Immunological characterization of samples from AD patients revealed elevated content of soluble A{beta} oligomers, detected by the conformation-dependent OC-antibody, whereas no immunoreactivity was detected in the normal samples. Intracellular injection of brain extracts from control patients failed to trigger detectable changes in intracellular Ca2+. Conversely, brain extracts from AD patients triggered Ca2+ events consisting of local and global Ca2+ fluorescent transients rising within few seconds after injection and persisting for several seconds. Pre-incubation of brain extracts with the conformation specific OC antibody completely suppressed brain extract ability to trigger cytosolic Ca2+ events. Comparison of the elementary events triggered by brain extracts and synthetic A{beta}42 oligomer showed comparable temporal evolution and amplitudes to events triggered by direct injection of IP3. Moreover, bath application of caffeine reversibly inhibited local and global Ca2+ signals in all the samples confirming the involvement of Ca2+ release from the ER. Analysis of the recorded Ca2+ fluorescence signals by computational modeling allowed quantification of the IP3 and Ca2+ generated by each sample. The model further shows that the abnormal increase of Ca2+ and IP3 may affect mitochondrial bioenergetics. These results, supports the hypothesis that endogenous amyloid oligomer contained in neurons of AD-affected brains may represent the toxic agents responsible for neurons malfunctioning and death, associated with the disruption of neuronal Ca2+ homeostasis.

cell biology↗

bioRxiv2022

Aβ42 and Aβ40 oligomers form transient and persistent pores with different time evolutions and toxicities

In Alzheimers disease (AD), formation of harmful self-gating pores formed by the insertion of amyloid beta oligomers (A{beta}Os) into the plasma membrane have been shown to cause disruption of Ca2+ homeostasis, leading to neuronal malfunctioning and degeneration. Among different isoforms, the most studied A{beta}40 and A{beta}42 are also believed to be the most toxic ones. Using single channel imaging, we show that both isoforms can form functionally distinct populations of Ca2+ permeable pores, we named transient and persistent pores. The transient pores could be seen only for a few tens of milliseconds, while persistent pores can be observed for more than an hour. However, while the Ca2+-toxicity of pores formed by A{beta}42Os tend to increase over time by displaying higher open probability and larger Ca2+ permeability, pores formed by A{beta}40Os show opposite time dependent behavior. We conclude that although both isoforms can form Ca2+ permeable pores in the cells plasma membrane, pores due to A{beta}42Os display worsening Ca2+ toxicity over time.

cell biology↗

bioRxiv2022

Upregulated Ca2+ release from the endoplasmic reticulum leads to impaired presynaptic function in Alzheimer's disease

Neurotransmitter release from presynaptic terminals is primarily regulated by rapid Ca2+ influx through membrane-resident voltage-gated Ca2+ channels (VGCCs). Also, accumulating evidence indicates that the endoplasmic reticulum (ER) is extensively present in axonal terminals of neurons and plays a modulatory role in synaptic transmission by regulating Ca2+ levels. Alzheimers disease (AD) is marked by enhanced Ca2+ release from the ER and downregulation of Ca2+ buffering proteins. However, the precise consequence of impaired Ca2+ signalling within the vicinity of VGCCs (active zone (AZ)) on exocytosis is poorly understood. Here, we perform in-silico experiments of intracellular Ca2+ signalling and exocytosis in a detailed biophysical model of hippocampal synapses to investigate the effect of aberrant Ca2+ signalling on neurotransmitter release in AD. Our model predicts that enhanced Ca2+ release from the ER increases the probability of neurotransmitter release in AD. Moreover, over very short timescales (30-60 msec), the model exhibits activity-dependent and enhanced short-term plasticity in AD, indicating neuronal hyperactivity--a hallmark of the disease. Similar to previous observations in AD animal models, our model reveals that during prolonged stimulation (~450 msec), pathological Ca2+ signalling increases depression and desynchronization with stimulus, causing affected synapses to operate unreliably. Overall, our work provides direct evidence in support of a crucial role played by altered Ca2+ homeostasis mediated by intracellular stores in AD.

neuroscience↗

bioRxiv2018

CellSpecks: A Software for Automated Detection and Analysis of Calcium Channels in Live Cells

To couple the fidelity of patch-clamp recording with a more high-throughput screening capability, we pioneered a novel approach to single channel recording that we named \"optical patch clamp\". By using highly-sensitive fluorescent Ca2+ indicator dyes in conjunction with total internal fluorescence microscopy techniques, we monitor Ca2+ flux through individual Ca2+-permeable channels. This approach provides information about channel gating analogous to patch-clamp recording at time resolution of ~ 2 ms, with the additional advantage of being massively parallel, providing simultaneous and independent recording from thousands of channels in native environment. However, manual analysis of the data generated by this technique presents severe challenges as a video recording can include many thousands of frames. To overcome this bottleneck, we developed an image processing and analysis framework called CellSpecks, capable of detecting and fully analyzing the kinetics of ion channels within a video sequence. By using a randomly generated synthetic data, we tested the ability of CellSpecks to rapidly and efficiently detect and analyze the activity of thousands of ion channels, including openings for a few milliseconds. Here, we report the use of CellSpecks for the analysis of experimental data acquired by imaging muscle nicotinic acetylcholine receptors and the Alzheimers disease-associated amyloid beta pores with multiconductance levels in the plasma membrane of Xenopus laevis oocytes. We show that CellSpecks can accurately and efficiently generate location maps, create raw and processed fluorescence time-traces, histograms of mean open times, mean close times, open probabilities, durations, and maximum amplitudes, and a channel chip showing the activity of all channels as a function of time. Although we specifically illustrate the application of CellSpecks for analyzing data from Ca2+ channels, it can be easily customized to analyze other spatially and temporally localized signals.

bioinformatics↗

bioRxiv2018

TraceSpecks: a software for automated idealization of noisy patch-clamp and imaging data

Experimental records of single molecules or ion channels from fluorescence microscopy and patch-clamp electrophysiology often include high-frequency noise and baseline fluctuations that are not generated by the system under investigation and have to be removed. More-over, multiple channels or conductance levels can be present at a time in the data that need to be quantified to accurately understand the behavior of the system. Manual procedures for removing these fluctuations and extracting conducting states or multiple channels are laborious, prone to subjective bias, and hinder the processing of often very large data-sets. We introduce a maximum likelihood formalism for separating signal from a noisy and drifting background such as fluorescence traces from imaging of elementary Ca2+ release events called puffs arising from clusters of channels and patch-clamp recordings of ion channels. Parameters such as the number of open channels or conducting states, noise level, and back-ground signal can all be optimized using the expectation-maximization (EM) algorithm. We implement our algorithm following the Baum-Welch approach to EM in the portable java language with a user-friendly graphical interface and test the algorithm on both synthetic and experimental data from patch-clamp electrophysiology of Ca2+ channels and fluorescence microscopy of a cluster of Ca2+ channels and Ca2+ channels with multiple conductance levels. The resulting software is accurate, fast, and provides detailed information usually not available through manual analysis. Options for visual inspection of the raw and processed data with key parameters, and exporting a range of statistics such as the mean open probabilities, mean open times, mean close times, and dwell time distributions for different number of channels open or conductance levels, amplitude distribution of all opening events, and number of transitions between different number of open channels or conducting levels in asci format with a single click are provided.

bioinformatics↗