Analysis of pressure-activated Piezo1 open and subconductance states at a single channel level.

Mechanically activated Piezo1 channels undergo transitions from closed to open-state in response to pressure and other mechanical stimuli. However, the molecular details of these mechanosensitive gating transitions are unknown. Here, we used cell-attached pressure-clamp recordings to acquire single channel data at steady-state conditions (where inactivation has settled down), at various pressures and voltages. Importantly, we identify and analyze subconductance states of the channel which were not reported before. Pressure-dependent activation of Piezo1 increases the occupancy of open and subconductance state at the expense of decreased occupancy of shut-states. No significant change in the mean open time of subconductance states was observed with increasing negative pipette pressure or with varying voltages (ranging from -40 to -100 mV). Using Markov-chain modeling, we identified a minimal four-states kinetic scheme, which recapitulates essential characteristics of the single channel data, including that of the subconductance level. This study advances our understanding of Piezo1-gating mechanism in response to discrete stimuli (such as pressure and voltage) and paves the path to develop cellular and tissue level models to predict Piezo1 function in various cell types.

Impaired θ-γ Coupling Indicates Inhibitory Dysfunction and Seizure Risk in a Dravet Syndrome Mouse Model.

Dravet syndrome (DS) is an epileptic encephalopathy that still lacks biomarkers for epileptogenesis and its treatment. Dysfunction of NaV1.1 sodium channels, which are chiefly expressed in inhibitory interneurons, explains the epileptic phenotype. Understanding the network effects of these cellular deficits may help predict epileptogenesis. Here, we studied θ-γ coupling as a potential marker for altered inhibitory functioning and epileptogenesis in a DS mouse model. We found that cortical θ-γ coupling was reduced in both male and female juvenile DS mice and persisted only if spontaneous seizures occurred. θ-γ Coupling was partly restored by cannabidiol (CBD). Locally disrupting NaV1.1 expression in the hippocampus or cortex yielded early attenuation of θ-γ coupling, which in the hippocampus associated with fast ripples, and which was replicated in a computational model when voltage-gated sodium currents were impaired in basket cells (BCs). Our results indicate attenuated θ-γ coupling as a promising early indicator of inhibitory dysfunction and seizure risk in DS.

The Critical Role of Spreading Depolarizations in Early Brain Injury: Consensus and Contention.

BACKGROUND: When a patient arrives in the emergency department following a stroke, a traumatic brain injury, or sudden cardiac arrest, there is no therapeutic drug available to help protect their jeopardized neurons. One crucial reason is that we have not identified the molecular mechanisms leading to electrical failure, neuronal swelling, and blood vessel constriction in newly injured gray matter. All three result from a process termed spreading depolarization (SD). Because we only partially understand SD, we lack molecular targets and biomarkers to help neurons survive after losing their blood flow and then undergoing recurrent SD. METHODS: In this review, we introduce SD as a single or recurring event, generated in gray matter following lost blood flow, which compromises the Na+/K+ pump. Electrical recovery from each SD event requires so much energy that neurons often die over minutes and hours following initial injury, independent of extracellular glutamate. RESULTS: We discuss how SD has been investigated with various pitfalls in numerous experimental preparations, how overtaxing the Na+/K+ ATPase elicits SD. Elevated K+ or glutamate are unlikely natural activators of SD. We then turn to the properties of SD itself, focusing on its initiation and propagation as well as on computer modeling. CONCLUSIONS: Finally, we summarize points of consensus and contention among the authors as well as where SD research may be heading. In an accompanying review, we critique the role of the glutamate excitotoxicity theory, how it has shaped SD research, and its questionable importance to the study of early brain injury as compared with SD theory.

Questioning Glutamate Excitotoxicity in Acute Brain Damage: The Importance of Spreading Depolarization.

BACKGROUND: Within 2 min of severe ischemia, spreading depolarization (SD) propagates like a wave through compromised gray matter of the higher brain. More SDs arise over hours in adjacent tissue, expanding the neuronal damage. This period represents a therapeutic window to inhibit SD and so reduce impending tissue injury. Yet most neuroscientists assume that the course of early brain injury can be explained by glutamate excitotoxicity, the concept that immediate glutamate release promotes early and downstream brain injury. There are many problems with glutamate release being the unseen culprit, the most practical being that the concept has yielded zero therapeutics over the past 30 years. But the basic science is also flawed, arising from dubious foundational observations beginning in the 1950s METHODS: Literature pertaining to excitotoxicity and to SD over the past 60 years is critiqued. RESULTS: Excitotoxicity theory centers on the immediate and excessive release of glutamate with resulting neuronal hyperexcitation. This instigates poststroke cascades with subsequent secondary neuronal injury. By contrast, SD theory argues that although SD evokes some brief glutamate release, acute neuronal damage and the subsequent cascade of injury to neurons are elicited by the metabolic stress of SD, not by excessive glutamate release. The challenge we present here is to find new clinical targets based on more informed basic science. This is motivated by the continuing failure by neuroscientists and by industry to develop drugs that can reduce brain injury following ischemic stroke, traumatic brain injury, or sudden cardiac arrest. One important step is to recognize that SD plays a central role in promoting early neuronal damage. We argue that uncovering the molecular biology of SD initiation and propagation is essential because ischemic neurons are usually not acutely injured unless SD propagates through them. The role of glutamate excitotoxicity theory and how it has shaped SD research is then addressed, followed by a critique of its fading relevance to the study of brain injury. CONCLUSIONS: Spreading depolarizations better account for the acute neuronal injury arising from brain ischemia than does the early and excessive release of glutamate.

On the origin of ultraslow spontaneous Na+ fluctuations in neurons of the neonatal forebrain.

Spontaneous neuronal and astrocytic activity in the neonate forebrain is believed to drive the maturation of individual cells and their integration into complex brain-region-specific networks. The previously reported forms include bursts of electrical activity and oscillations in intracellular Ca2+ concentration. Here, we use ratiometric Na+ imaging to demonstrate spontaneous fluctuations in the intracellular Na+ concentration of CA1 pyramidal neurons and astrocytes in tissue slices obtained from the hippocampus of mice at postnatal days 2-4 (P2-4). These occur at very low frequency (∼2/h), can last minutes with amplitudes up to several millimolar, and mostly disappear after the first postnatal week. To further investigate their mechanisms, we model a network consisting of pyramidal neurons and interneurons. Experimentally observed Na+ fluctuations are mimicked when GABAergic inhibition in the simulated network is made depolarizing. Both our experiments and computational model show that blocking voltage-gated Na+ channels or GABAergic signaling significantly diminish the neuronal Na+ fluctuations. On the other hand, blocking a variety of other ion channels, receptors, or transporters including glutamatergic pathways does not have significant effects. Our model also shows that the amplitude and duration of Na+ fluctuations decrease as we increase the strength of glial K+ uptake. Furthermore, neurons with smaller somatic volumes exhibit fluctuations with higher frequency and amplitude. As opposed to this, larger extracellular to intracellular volume ratio observed in neonatal brain exerts a dampening effect. Finally, our model predicts that these periods of spontaneous Na+ influx leave neonatal neuronal networks more vulnerable to seizure-like states when compared with mature brain.NEW & NOTEWORTHY Spontaneous activity in the neonate forebrain plays a key role in cell maturation and brain development. We report spontaneous, ultraslow, asynchronous fluctuations in the intracellular Na+ concentration of neurons and astrocytes. We show that this activity is not correlated with the previously reported synchronous neuronal population bursting or Ca2+ oscillations, both of which occur at much faster timescales. Furthermore, extracellular K+ concentration remains nearly constant. The spontaneous Na+ fluctuations disappear after the first postnatal week.

Mitochondrial dysfunction and role in spreading depolarization and seizure.

The effect of pathological phenomena such as epileptic seizures and spreading depolarization (SD) on mitochondria and the potential feedback of mitochondrial dysfunction into the dynamics of those phenomena are complex and difficult to study experimentally due to the simultaneous changes in many variables governing neuronal behavior. By combining a model that accounts for a wide range of neuronal behaviors including seizures, normoxic SD, and hypoxic SD (HSD), together with a detailed model of mitochondrial function and intracellular Ca2+ dynamics, we investigate mitochondrial dysfunction and its potential role in recovery of the neuron from seizures, HSD, and SD. Our results demonstrate that HSD leads to the collapse of mitochondrial membrane potential and cellular ATP levels that recover only when normal oxygen supply is restored. Mitochondrial organic phosphate and pH gradients determine the strength of the depolarization block during HSD and SD, how quickly the cell enters the depolarization block when the oxygen supply is disrupted or potassium in the bath solution is raised beyond the physiological value, and how fast the cell recovers from SD and HSD when normal potassium concentration and oxygen supply are restored. Although not as dramatic as phosphate and pH gradients, mitochondrial Ca2+ uptake has a similar effect on neuronal behavior during these conditions.

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, to our knowledge, 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 a time resolution of ∼2 ms with the additional advantage of being massively parallel, providing simultaneous and independent recording from thousands of channels in the native environment. However, manual analysis of the data generated by this technique presents severe challenges because 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 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 Alzheimer's disease-associated amyloid ß pores with multiconductance levels in the plasma membrane of Xenopus laevis oocytes. We show that CellSpecks can accurately and efficiently generate location maps and create raw and processed fluorescence time traces; histograms of mean open times, mean close times, open probabilities, durations, and maximal 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.

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. Moreover, 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 likely to hinder the processing of often very large data sets. We introduce a maximal 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 background signal can all be optimized using the expectation-maximization algorithm. We implement our algorithm following the Baum-Welch approach to expectation-maximization in the portable Java language with a user-friendly graphical interface and test the algorithm on both synthetic and experimental data from the 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 are provided, in addition to a range of statistics such as the mean open probabilities, mean open times, mean close times, 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.

The role of glutamate in neuronal ion homeostasis: A case study of spreading depolarization.

Simultaneous changes in ion concentrations, glutamate, and cell volume together with exchange of matter between cell network and vasculature are ubiquitous in numerous brain pathologies. A complete understanding of pathological conditions as well as normal brain function, therefore, hinges on elucidating the molecular and cellular pathways involved in these mostly interdependent variations. In this paper, we develop the first computational framework that combines the Hodgkin-Huxley type spiking dynamics, dynamic ion concentrations and glutamate homeostasis, neuronal and astroglial volume changes, and ion exchange with vasculature into a comprehensive model to elucidate the role of glutamate uptake in the dynamics of spreading depolarization (SD)-the electrophysiological event underlying numerous pathologies including migraine, ischemic stroke, aneurysmal subarachnoid hemorrhage, intracerebral hematoma, and trauma. We are particularly interested in investigating the role of glutamate in the duration and termination of SD caused by K+ perfusion and oxygen-glucose deprivation. Our results demonstrate that glutamate signaling plays a key role in the dynamics of SD, and that impaired glutamate uptake leads to recovery failure of neurons from SD. We confirm predictions from our model experimentally by showing that inhibiting astrocytic glutamate uptake using TFB-TBOA nearly quadruples the duration of SD in layers 2-3 of visual cortical slices from juvenile rats. The model equations are either derived purely from first physical principles of electroneutrality, osmosis, and conservation of particles or a combination of these principles and known physiological facts. Accordingly, we claim that our approach can be used as a future guide to investigate the role of glutamate, ion concentrations, and dynamics cell volume in other brain pathologies and normal brain function.

Reduced cooperativity of voltage-gated sodium channels in the hippocampal interneurons of an aged mouse model of Alzheimer's disease.

Beta amyloid (A[Formula: see text] ) associated with Alzheimer's disease (AD) leads to abnormal behavior in inhibitory neurons, resulting in hyperactive neuronal networks, epileptiform behavior, disrupted gamma rhythms, and aberrant synaptic plasticity. Previously, we used a dual modeling-experimental approach to explain several observations, including failure to reliably produce action potentials (APs), smaller AP amplitudes, higher resting membrane potential, and higher membrane depolarization in response to a range of stimuli in hippocampal inhibitory neurons from 12- to 16-month-old female AP Pswe/PSEN1DeltaE9 (APdE9) AD mice as compared to age-matched non-transgenic (NTG) mice. Our experimental results also showed that AP initiation in interneurons from APdE9 mice are significantly different from that of NTG mice. APs in interneurons from NTG mice are characterized by abrupt onset and an upstroke that is much steeper and occurs with larger variability as compared to cells from APdE9 mice. The phase plot (the rate of change of membrane potential versus the instantaneous membrane potential) of APs produced by interneurons from APdE9 mice shows a biphasic behavior, whereas that from NTG mice shows a monophasic behavior. Here we show that using the classic Hodgkin-Huxley (HH) formalism for the gating of voltage-gated sodium channels (VGSCs) in a single-compartment neuron, we cannot reproduce these features, and a model that takes into account a cooperative activation of VGSCs is needed. We also argue that considering a realistic multi-compartment neuron where the kinetics of VGSC is modeled by HH formalism, as done in the past, would not explain our observations when APs from both NTG and APdE9 mice are considered simultaneously. We further show that VGSCs in interneurons from APdE9 mice exhibit significantly lower cooperativity in their activation as compared to those from NTG mice.

Large extracellular space leads to neuronal susceptibility to ischemic injury in a Na+/K+ pumps-dependent manner.

The extent of anoxic depolarization (AD), the initial electrophysiological event during ischemia, determines the degree of brain region-specific neuronal damage. Neurons in higher brain regions exhibiting nonreversible, strong AD are more susceptible to ischemic injury as compared to cells in lower brain regions that exhibit reversible, weak AD. While the contrasting ADs in different brain regions in response to oxygen-glucose deprivation (OGD) is well established, the mechanism leading to such differences is not clear. Here we use computational modeling to elucidate the mechanism behind the brain region-specific recovery from AD. Our extended Hodgkin-Huxley (HH) framework consisting of neural spiking dynamics, processes of ion accumulation, and ion homeostatic mechanisms unveils that glial-vascular K(+) clearance and Na(+)/K(+)-exchange pumps are key to the cell's recovery from AD. Our phase space analysis reveals that the large extracellular space in the upper brain regions leads to impaired Na(+)/K(+)-exchange pumps so that they function at lower than normal capacity and are unable to bring the cell out of AD after oxygen and glucose is restored.

The Role of Cell Volume in the Dynamics of Seizure, Spreading Depression, and Anoxic Depolarization.

Cell volume changes are ubiquitous in normal and pathological activity of the brain. Nevertheless, we know little of how cell volume affects neuronal dynamics. We here performed the first detailed study of the effects of cell volume on neuronal dynamics. By incorporating cell swelling together with dynamic ion concentrations and oxygen supply into Hodgkin-Huxley type spiking dynamics, we demonstrate the spontaneous transition between epileptic seizure and spreading depression states as the cell swells and contracts in response to changes in osmotic pressure. Our use of volume as an order parameter further revealed a dynamical definition for the experimentally described physiological ceiling that separates seizure from spreading depression, as well as predicted a second ceiling that demarcates spreading depression from anoxic depolarization. Our model highlights the neuroprotective role of glial K buffering against seizures and spreading depression, and provides novel insights into anoxic depolarization and the relevant cell swelling during ischemia. We argue that the dynamics of seizures, spreading depression, and anoxic depolarization lie along a continuum of the repertoire of the neuron membrane that can be understood only when the dynamic ion concentrations, oxygen homeostasis,and cell swelling in response to osmotic pressure are taken into consideration. Our results demonstrate the feasibility of a unified framework for a wide range of neuronal behaviors that may be of substantial importance in the understanding of and potentially developing universal intervention strategies for these pathological states.

Analyzing and Quantifying the Gain-of-Function Enhancement of IP3 Receptor Gating by Familial Alzheimer's Disease-Causing Mutants in Presenilins.

Familial Alzheimer's disease (FAD)-causing mutant presenilins (PS) interact with inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) Ca(2+) release channels resulting in enhanced IP3R channel gating in an amyloid beta (Aß) production-independent manner. This gain-of-function enhancement of IP3R activity is considered to be the main reason behind the upregulation of intracellular Ca(2+) signaling in the presence of optimal and suboptimal stimuli and spontaneous Ca(2+) signals observed in cells expressing mutant PS. In this paper, we employed computational modeling of single IP3R channel activity records obtained under optimal Ca(2+) and multiple IP3 concentrations to gain deeper insights into the enhancement of IP3R function. We found that in addition to the high occupancy of the high-activity (H) mode and the low occupancy of the low-activity (L) mode, IP3R in FAD-causing mutant PS-expressing cells exhibits significantly longer mean life-time for the H mode and shorter life-time for the L mode, leading to shorter mean close-time and hence high open probability of the channel in comparison to IP3R in cells expressing wild-type PS. The model is then used to extrapolate the behavior of the channel to a wide range of IP3 and Ca(2+) concentrations and quantify the sensitivity of IP3R to its two ligands. We show that the gain-of-function enhancement is sensitive to both IP3 and Ca(2+) and that very small amount of IP3 is required to stimulate IP3R channels in the presence of FAD-causing mutant PS to the same level of activity as channels in control cells stimulated by significantly higher IP3 concentrations. We further demonstrate with simulations that the relatively longer time spent by IP3R in the H mode leads to the observed higher frequency of local Ca(2+) signals, which can account for the more frequent global Ca(2+) signals observed, while the enhanced activity of the channel at extremely low ligand concentrations will lead to spontaneous Ca(2+) signals in cells expressing FAD-causing mutant PS.

Mode switching of Inositol 1,4,5-trisphosphate receptor channel shapes the Spatiotemporal scales of Ca2+ signals.

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) channel is crucial for the generation and modulation of highly specific intracellular Ca2+ signals performing numerous functions in animal cells. However, the single channel behavior during Ca2+ signals of different spatiotemporal scales is not well understood. To elucidate the correlation between the gating dynamics of single InsP3Rs and spatiotemporal Ca2+ patterns, we simulate a cluster of InsP3Rs under varying ligand concentrations and extract comprehensive gating statistics of all channels during events of different sizes and durations. Our results show that channels gating predominantly in the low activity mode with negligible occupancy of intermediate and high modes leads to single channel Ca2+ release event blips. Increasing occupancies of intermediate and high modes results in events with increasing size. When the channel has more than 50% probability of gating in the intermediate and high modes, the cluster generates very large puffs that would most likely result in global Ca2+ signals. The size, duration and frequency of Ca2+ signals all increase linearly with the total probability of channel gating in the intermediate and high modes. To our knowledge, this is the first study that quantitatively relates the modal characteristics of InsP3R to the shaping of different spatiotemporal scales of Ca2+ signals.

Unification of neuronal spikes, seizures, and spreading depression.

The pathological phenomena of seizures and spreading depression have long been considered separate physiological events in the brain. By incorporating conservation of particles and charge, and accounting for the energy required to restore ionic gradients, we extend the classic Hodgkin-Huxley formalism to uncover a unification of neuronal membrane dynamics. By examining the dynamics as a function of potassium and oxygen, we now account for a wide range of neuronal activities, from spikes to seizures, spreading depression (whether high potassium or hypoxia induced), mixed seizure and spreading depression states, and the terminal anoxic "wave of death." Such a unified framework demonstrates that all of these dynamics lie along a continuum of the repertoire of the neuron membrane. Our results demonstrate that unified frameworks for neuronal dynamics are feasible, can be achieved using existing biological structures and universal physical conservation principles, and may be of substantial importance in enabling our understanding of brain activity and in the control of pathological states.

Oxygen and seizure dynamics: II. Computational modeling.

Electrophysiological recordings show intense neuronal firing during epileptic seizures leading to enhanced energy consumption. However, the relationship between oxygen metabolism and seizure patterns has not been well studied. Recent studies have developed fast and quantitative techniques to measure oxygen microdomain concentration during seizure events. In this article, we develop a biophysical model that accounts for these experimental observations. The model is an extension of the Hodgkin-Huxley formalism and includes the neuronal microenvironment dynamics of sodium, potassium, and oxygen concentrations. Our model accounts for metabolic energy consumption during and following seizure events. We can further account for the experimental observation that hypoxia can induce seizures, with seizures occurring only within a narrow range of tissue oxygen pressure. We also reproduce the interplay between excitatory and inhibitory neurons seen in experiments, accounting for the different oxygen levels observed during seizures in excitatory vs. inhibitory cell layers. Our findings offer a more comprehensive understanding of the complex interrelationship among seizures, ion dynamics, and energy metabolism.

Bidirectional dysregulation of synaptic glutamate signaling after transient metabolic failure.

Ischemia leads to a severe dysregulation of glutamate homeostasis and excitotoxic cell damage in the brain. Shorter episodes of energy depletion, for instance during peri-infarct depolarizations, can also acutely perturb glutamate signaling. It is less clear if such episodes of metabolic failure also have persistent effects on glutamate signaling and how the relevant mechanisms such as glutamate release and uptake are differentially affected. We modelled acute and transient metabolic failure by using a chemical ischemia protocol and analyzed its effect on glutamatergic synaptic transmission and extracellular glutamate signals by electrophysiology and multiphoton imaging, respectively, in the hippocampus. Our experiments uncover a duration-dependent bidirectional dysregulation of glutamate signaling. Whereas short chemical ischemia induces a lasting potentiation of presynaptic glutamate release and synaptic transmission, longer episodes result in a persistent postsynaptic failure of synaptic transmission. We also observed unexpected differences in the vulnerability of the investigated cellular mechanisms. Axonal action potential firing and glutamate uptake were unexpectedly resilient compared to postsynaptic cells, which overall were most vulnerable to acute and transient metabolic stress. We conclude that even short perturbations of energy supply lead to a lasting potentiation of synaptic glutamate release, which may increase glutamate excitotoxicity well beyond the metabolic incident.

Systematic Analysis of Biological Processes Reveals Gene Co-expression Modules Driving Pathway Dysregulation in Alzheimer's Disease.

Alzheimer's disease (AD) manifests as a complex systems pathology with intricate interplay among various genes and biological processes. Traditional differential gene expression (DEG) analysis, while commonly employed to characterize AD-driven perturbations, does not sufficiently capture the full spectrum of underlying biological processes. Utilizing single-nucleus RNA-sequencing data from postmortem brain samples across key regions-middle temporal gyrus, superior frontal gyrus, and entorhinal cortex-we provide a comprehensive systematic analysis of disrupted processes in AD. We go beyond the DEG-centric analysis by integrating pathway activity analysis with weighted gene co-expression patterns to comprehensively map gene interconnectivity, identifying region- and cell-type-specific drivers of biological processes associated with AD. Our analysis reveals profound modular heterogeneity in neurons and glia as well as extensive AD-related functional disruptions. Co-expression networks highlighted the extended involvement of astrocytes and microglia in biological processes beyond neuroinflammation, such as calcium homeostasis, glutamate regulation, lipid metabolism, vesicle-mediated transport, and TOR signaling. We find limited representation of DEGs within dysregulated pathways across neurons and glial cells, suggesting that differential gene expression alone may not adequately represent the disease complexity. Further dissection of inferred gene modules revealed distinct dynamics of hub DEGs in neurons versus glia, suggesting that DEGs exert more impact on neurons compared to glial cells in driving modular dysregulations underlying perturbed biological processes. Interestingly, we observe an overall downregulation of astrocyte and microglia modules across all brain regions in AD, indicating a prevailing trend of functional repression in glial cells across these regions. Notable genes from the CALM and HSP90 families emerged as hub genes across neuronal modules in all brain regions, suggesting conserved roles as drivers of synaptic dysfunction in AD. Our findings demonstrate the importance of an integrated, systems-oriented approach combining pathway and network analysis to comprehensively understand the cell-type-specific roles of genes in AD-related biological processes.

Systematic Analysis of Biological Processes Reveals Gene Co-expression Modules Driving Pathway Dysregulation in Alzheimer's Disease.

Alzheimer's disease (AD) manifests as a complex systems pathology with intricate interplay among various genes and biological processes. Traditional differential gene expression (DEG) analysis, while commonly employed to characterize AD-driven perturbations, does not sufficiently capture the full spectrum of underlying biological processes. Utilizing single-nucleus RNA-sequencing data from postmortem brain samples across key regions-middle temporal gyrus, superior frontal gyrus, and entorhinal cortex-we provide a comprehensive systematic analysis of disrupted processes in AD. We go beyond the DEG-centric analysis by integrating pathway activity analysis with weighted gene co-expression patterns to comprehensively map gene interconnectivity, identifying region- and cell-type-specific drivers of biological processes associated with AD. Our analysis reveals profound modular heterogeneity in neurons and glia as well as extensive AD-related functional disruptions. Co-expression networks highlighted the extended involvement of astrocytes and microglia in biological processes beyond neuroinflammation, such as calcium homeostasis, glutamate regulation, lipid metabolism, vesicle-mediated transport, and TOR signaling. We find limited representation of DEGs within dysregulated pathways across neurons and glial cells, indicating that differential gene expression alone may not adequately represent the disease complexity. Further dissection of inferred gene modules revealed distinct dynamics of hub DEGs in neurons versus glia, highlighting the differential impact of DEGs on neurons compared to glial cells in driving modular dysregulations underlying perturbed biological processes. Interestingly, we note an overall downregulation of both astrocyte and microglia modules in AD across all brain regions, suggesting a prevailing trend of functional repression in glial cells across these regions. Notable genes, including those of the CALM and HSP90 family genes emerged as hub genes across neuronal modules in all brain regions, indicating conserved roles as drivers of synaptic dysfunction in AD. Our findings demonstrate the importance of an integrated, systems-oriented approach combining pathway and network analysis for a comprehensive understanding of the cell-type-specific roles of genes in AD-related biological processes.

Automated maximum likelihood separation of signal from baseline in noisy quantal data.

Data recordings often include high-frequency noise and baseline fluctuations that are not generated by the system under investigation, which need to be removed before analyzing the signal for the system's behavior. In the absence of an automated method, experimentalists fall back on manual procedures for removing these fluctuations, which can be laborious and prone to subjective bias. We introduce a maximum likelihood formalism for separating signal from a drifting baseline plus noise, when the signal takes on integer multiples of some value, as in ion channel patch-clamp current traces. Parameters such as the quantal step size (e.g., current passing through a single channel), noise amplitude, and baseline drift rate can all be optimized automatically using the expectation-maximization algorithm, taking the number of open channels (or molecules in the on-state) at each time point as a hidden variable. Our goal here is to reconstruct the signal, not model the (possibly highly complex) underlying system dynamics. Thus, our likelihood function is independent of those dynamics. This may be thought of as restricting to the simplest possible hidden Markov model for the underlying channel current, in which successive measurements of the state of the channel(s) are independent. The resulting method is comparable to an experienced human in terms of results, but much faster. FORTRAN 90, C, R, and JAVA codes that implement the algorithm are available for download from our website.