NeuroActivityToolkit-Toolbox for Quantitative Analysis of Miniature Fluorescent Microscopy Data.

The visualization of neuronal activity in vivo is an urgent task in modern neuroscience. It allows neurobiologists to obtain a large amount of information about neuronal network architecture and connections between neurons. The miniscope technique might help to determine changes that occurred in the network due to external stimuli and various conditions: processes of learning, stress, epileptic seizures and neurodegenerative diseases. Furthermore, using the miniscope method, functional changes in the early stages of such disorders could be detected. The miniscope has become a modern approach for recording hundreds to thousands of neurons simultaneously in a certain brain area of a freely behaving animal. Nevertheless, the analysis and interpretation of the large recorded data is still a nontrivial task. There are a few well-working algorithms for miniscope data preprocessing and calcium trace extraction. However, software for further high-level quantitative analysis of neuronal calcium signals is not publicly available. NeuroActivityToolkit is a toolbox that provides diverse statistical metrics calculation, reflecting the neuronal network properties such as the number of neuronal activations per minute, amount of simultaneously co-active neurons, etc. In addition, the module for analyzing neuronal pairwise correlations is implemented. Moreover, one can visualize and characterize neuronal network states and detect changes in 2D coordinates using PCA analysis. This toolbox, which is deposited in a public software repository, is accompanied by a detailed tutorial and is highly valuable for the statistical interpretation of miniscope data in a wide range of experimental tasks.

Positive Allosteric Modulators of SERCA Pump Restore Dendritic Spines and Rescue Long-Term Potentiation Defects in Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is a neurodegenerative disorder that affects memory formation and storage processes. Dysregulated neuronal calcium (Ca2+) has been identified as one of the key pathogenic events in AD, and it has been suggested that pharmacological agents that stabilize Ca2+ neuronal signaling can act as disease-modifying agents in AD. In previous studies, we demonstrated that positive allosteric regulators (PAMs) of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump might act as such Ca2+-stabilizing agents and exhibit neuroprotective properties. In the present study, we evaluated effects of a set of novel SERCA PAM agents on the rate of Ca2+ extraction from the cytoplasm of the HEK293T cell line, on morphometric parameters of dendritic spines of primary hippocampal neurons in normal conditions and in conditions of amyloid toxicity, and on long-term potentiation in slices derived from 5xFAD transgenic mice modeling AD. Several SERCA PAM compounds demonstrated neuroprotective properties, and the compound NDC-9009 showed the best results. The findings in this study support the hypothesis that the SERCA pump is a potential therapeutic target for AD treatment and that NDC-9009 is a promising lead molecule to be used in the development of disease-modifying agents for AD.

SpineTool is an open-source software for analysis of morphology of dendritic spines.

Dendritic spines form most excitatory synaptic inputs in neurons and these spines are altered in many neurodevelopmental and neurodegenerative disorders. Reliable methods to assess and quantify dendritic spines morphology are needed, but most existing methods are subjective and labor intensive. To solve this problem, we developed an open-source software that allows segmentation of dendritic spines from 3D images, extraction of their key morphological features, and their classification and clustering. Instead of commonly used spine descriptors based on numerical metrics we used chord length distribution histogram (CLDH) approach. CLDH method depends on distribution of lengths of chords randomly generated within dendritic spines volume. To achieve less biased analysis, we developed a classification procedure that uses machine-learning algorithm based on experts' consensus and machine-guided clustering tool. These approaches to unbiased and automated measurements, classification and clustering of synaptic spines that we developed should provide a useful resource for a variety of neuroscience and neurodegenerative research applications.

Expansion Microscopy Application for Calcium Protein Clustering Imaging in Cells and Brain Tissues.

Many biological studies require high-resolution imaging and subsequent analysis of cell organelles and molecules. Some membrane proteins form tight clusters, and this process is directly linked to their function. In most studies, these small protein clusters have been investigated by total internal reflection fluorescence (TIRF) microscopy, which enables imaging with high spatial resolution within 100 nm of the membrane surface. Recently developed expansion microscopy (ExM) makes it possible to achieve nanometer resolution using a conventional fluorescence microscope by physically expanding the sample. In this article, we describe implementation of ExM for imaging of protein clusters formed by the endoplasmic reticulum (ER) calcium sensor protein STIM1. This protein translocates during ER store depletion and forms clusters that support contact with plasma membrane (PM) calcium-channel proteins. ER calcium channels such as the type 1 inositol triphosphate receptor (IP3R) also form clusters, but their investigation by TIRF microscopy is impossible due to the large distance from the PM. In this article, we demonstrate how to investigate IP3R clustering using ExM in hippocampal brain tissues. We compare IP3R clustering in the CA1 area of the hippocampus of wild-type and 5xFAD Alzheimer's disease model mice. To facilitate future applications, we describe experimental protocols and image processing guidelines for application of ExM to membrane and ER protein clustering studies in cultured cells and brain tissues. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Expansion microscopy application for protein cluster visualization in cells Alternate Protocol: Expansion microscopy application for protein cluster visualization in brain tissues Basic Protocol 2: Protein cluster analysis of expansion microscopy images using ImageJ and Icy software.

Cytoskeleton Protein EB3 Contributes to Dendritic Spines Enlargement and Enhances Their Resilience to Toxic Effects of Beta-Amyloid.

EB3 protein is expressed abundantly in the nervous system and transiently enters the dendritic spines at the tip of the growing microtubule, which leads to spine enlargement. Nevertheless, the role of dynamic microtubules, and particularly EB3 protein, in synapse function is still elusive. By manipulating the EB3 expression level, we have shown that this protein is required for a normal dendritogenesis. Nonetheless, EB3 overexpression also reduces hippocampal neurons dendritic branching and total dendritic length. This effect likely occurs due to the speeding neuronal development cycle from dendrite outgrowth to the step when dendritic spines are forming. Implementing direct morphometric characterization of dendritic spines, we showed that EB3 overexpression leads to a dramatic increase in the dendritic spine head area. EB3 knockout oppositely reduces spine head area and increases spine neck length and spine neck/spine length ratio. The same effect is observed in conditions of amyloid-beta toxicity, modeling Alzheimer`s disease. Neck elongation is supposed to be a common detrimental effect on the spine's shape, which makes them biochemically and electrically less connected to the dendrite. EB3 also potentiates the formation of presynaptic protein Synapsin clusters and CaMKII-alpha preferential localization in spines rather than in dendrites of hippocampal neurons, while its downregulation has an opposite effect and reduces the size of presynaptic protein clusters Synapsin and PSD95. EB3's role in spine development and maturation determines its neuroprotective effect. EB3 overexpression makes dendritic spines resilient to amyloid-beta toxicity, restores altered PSD95 clustering, and reduces CaMKII-alpha localization in spines observed in this pathological state.

Dendritic Spines Shape Analysis-Classification or Clusterization? Perspective.

Dendritic spines are small protrusions from the dendrite membrane, where contact with neighboring axons is formed in order to receive synaptic input. Changes in size, shape, and density of synaptic spines are associated with learning and memory, and observed after drug abuse in a variety of neurodegenerative, neurodevelopmental, and psychiatric disorders. Due to the preeminent importance of synaptic spines, there have been major efforts into developing techniques that enable visualization and analysis of dendritic spines in cultured neurons, in fixed slices and in intact brain tissue. The classification of synaptic spines into predefined morphological groups is a standard approach in neuroscience research, where spines are divided into fixed categories such as thin, mushroom, and stubby subclasses. This study examines accumulated evidence that supports the existence of dendritic spine shapes as a continuum rather than separated classes. Using new approaches and software tools we reflect on complex dendritic spine shapes, positing that understanding of their highly dynamic nature is required to perform analysis of their morphology. The study discusses and compares recently developed algorithms that rely on clusterization rather than classification, therefore enabling new levels of spine shape analysis. We reason that improved methods of analysis may help to investigate a link between dendritic spine shape and its function, facilitating future studies of learning and memory as well as studies of brain disorders.

Dysregulation of Intracellular Calcium Signaling in Alzheimer's Disease.

SIGNIFICANCE: Calcium (Ca2+) hypothesis of Alzheimer's disease (AD) gains popularity. It points to new signaling pathways that may underlie AD pathogenesis. Based on calcium hypothesis, novel targets for the development of potential AD therapies are identified. Recent Advances: Recently, the key role of neuronal store-operated calcium entry (nSOCE) in the development of AD has been described. Correct regulation of nSOCE is necessary for the stability of postsynaptic contacts to preserve the memory formation. Molecular identity of hippocampal nSOCE is defined. Perspective nSOCE-activating molecule, prototype of future anti-AD drugs, is described. CRITICAL ISSUES: Endoplasmic reticulum Ca2+ overload happens in many but not in all AD models. The nSOCE targeting therapy described in this review may not be universally applicable. FUTURE DIRECTIONS: There is a need to determine whether AD is a syndrome with one critical signaling pathway that initiates pathology, or it is a disorder with many different signaling pathways that are disrupted simultaneously or one after each other. It is necessary to validate applicability of nSOCE-activating therapy for the development of anti-AD medication. There is an experimental correlation between downregulated nSOCE and disrupted postsynaptic contacts in AD mouse models. Signaling mechanisms downstream of nSOCE which are responsible for the regulation of stability of postsynaptic contacts have to be discovered. That will bring new targets for the development of AD-preventing therapies. Antioxid. Redox Signal. 29, 1176-1188.

Calcium signaling and molecular mechanisms underlying neurodegenerative diseases.

Calcium (Ca2+) is a ubiquitous second messenger that regulates various activities in eukaryotic cells. Especially important role calcium plays in excitable cells. Neurons require extremely precise spatial-temporal control of calcium-dependent processes because they regulate such vital functions as synaptic plasticity. Recent evidence indicates that neuronal calcium signaling is abnormal in many of neurodegenerative disorders such as Alzheimer's disease (AD), Huntington's disease (HD) and Parkinson's disease (PD). These diseases represent a major medical, social, financial and scientific problem, but despite enormous research efforts, they are still incurable and only symptomatic relief drugs are available. Thus, new approaches and targets are needed. This review highlight neuronal calcium-signaling abnormalities in these diseases, with particular emphasis on the role of neuronal store-operated Ca2+ entry (SOCE) pathway and its potential relevance as a therapeutic target for treatment of neurodegeneration.

Stim2-Eb3 Association and Morphology of Dendritic Spines in Hippocampal Neurons.

Mushroom spines form strong synaptic contacts and are essential for memory storage. We have previously demonstrated that neuronal store-operated calcium entry (nSOC) in hippocampal neurons is regulated by STIM2 protein. This pathway plays a key role in stability of mushroom spines and is compromised in different mice models of Alzheimer's disease (AD). Actin was thought to be the sole cytoskeleton compartment presented in dendritic spines, however, recent studies demonstrated that dynamic microtubules with EB3 capped plus-ends transiently enter spines. We showed that STIM2 forms an endoplasmic reticulum (ER) Ca2+ -dependent complex with EB3 via Ser-x-Ile-Pro aminoacid motif and that disruption of STIM2-EB3 interaction resulted in loss of mushroom spines in hippocampal neurons. Overexpression of EB3 causes increase of mushroom spines fraction and is able to restore their deficiency in hippocampal neurons obtained from PS1-M146V-KI AD mouse model. STIM2 overexpression failed to restore mushroom dendritic spines after EB3 knockdown, while in contrast EB3 overexpression rescued loss of mushroom spines resulting from STIM2 depletion. We propose that EB3 is involved in regulation of dendritic spines morphology, in part due to its association with STIM2, and that modulation of EB3 expression is a potential way to overcome synaptic loss during AD.

Dysregulation of neuronal calcium homeostasis in Alzheimer's disease - A therapeutic opportunity?

Alzheimer's disease (AD) is the disease of lost memories. Synaptic loss is a major reason for memory defects in AD. Signaling pathways involved in memory loss in AD are under intense investigation. The role of deranged neuronal calcium (Ca2+) signaling in synaptic loss in AD is described in this review. Familial AD (FAD) mutations in presenilins are linked directly with synaptic Ca2+ signaling abnormalities, most likely by affecting endoplasmic reticulum (ER) Ca2+ leak function of presenilins. Excessive ER Ca2+ release via type 2 ryanodine receptors (RyanR2) is observed in AD spines due to increase in expression and function of RyanR2. Store-operated Ca2+ entry (nSOC) pathway is disrupted in AD spines due to downregulation of STIM2 protein. Because of these Ca2+ signaling abnormalities, a balance in activities of Ca2+-calmodulin-dependent kinase II (CaMKII) and Ca2+-dependent phosphatase calcineurin (CaN) is shifted at the synapse, tilting a balance between long-term potentiation (LTP) and long-term depression (LTD) synaptic mechanisms. As a result, synapses are weakened and eliminated in AD brains by LTD mechanism, causing memory loss. Targeting synaptic calcium signaling pathways offers opportunity for development of AD therapeutic agents.

Store-Operated Calcium Channel Complex in Postsynaptic Spines: A New Therapeutic Target for Alzheimer's Disease Treatment.

Mushroom dendritic spine structures are essential for memory storage and the loss of mushroom spines may explain memory defects in aging and Alzheimer's disease (AD). The stability of mushroom spines depends on stromal interaction molecule 2 (STIM2)-mediated neuronal-store-operated Ca2+ influx (nSOC) pathway, which is compromised in AD mouse models, in aging neurons, and in sporadic AD patients. Here, we demonstrate that the Transient Receptor Potential Canonical 6 (TRPC6) and Orai2 channels form a STIM2-regulated nSOC Ca2+ channel complex in hippocampal mushroom spines. We further demonstrate that a known TRPC6 activator, hyperforin, and a novel nSOC positive modulator, NSN21778 (NSN), can stimulate activity of nSOC pathway in the spines and rescue mushroom spine loss in both presenilin and APP knock-in mouse models of AD. We further show that NSN rescues hippocampal long-term potentiation impairment in APP knock-in mouse model. We conclude that the STIM2-regulated TRPC6/Orai2 nSOC channel complex in dendritic mushroom spines is a new therapeutic target for the treatment of memory loss in aging and AD and that NSN is a potential candidate molecule for therapeutic intervention in brain aging and AD. SIGNIFICANCE STATEMENT: Mushroom dendritic spine structures are essential for memory storage and the loss of mushroom spines may explain memory defects in Alzheimer's disease (AD). This study demonstrated that Transient Receptor Potential Canonical 6 (TRPC6) and Orai2 form stromal interaction molecule 2 (STIM2)-regulated neuronal-store-operated Ca2+ influx (nSOC) channel complex in hippocampal synapse and the resulting Ca2+ influx is critical for long-term maintenance of mushroom spines in hippocampal neurons. A novel nSOC-positive modulator, NSN21778 (NSN), rescues mushroom spine loss and synaptic plasticity impairment in AD mice models. The TRPC6/Orai2 nSOC channel complex is a new therapeutic target and NSN is a potential candidate molecule for therapeutic intervention in brain aging and AD.

Neuronal Store-Operated Calcium Entry and Mushroom Spine Loss in Amyloid Precursor Protein Knock-In Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common reason for elderly dementia in the world. We proposed that memory loss in AD is related to destabilization of mushroom postsynaptic spines involved in long-term memory storage. We demonstrated previously that stromal interaction molecule 2 (STIM2)-regulated neuronal store-operated calcium entry (nSOC) in postsynaptic spines play a key role in stability of mushroom spines by maintaining activity of synaptic Ca(2+)/calmodulin kinase II (CaMKII). Furthermore, we demonstrated previously that the STIM2-nSOC-CaMKII pathway is downregulated in presenilin 1 M146V knock-in (PS1-M146V KI) mouse model of AD, leading to loss of hippocampal mushroom spines in this model. In the present study, we demonstrate that hippocampal mushroom postsynaptic spines are also lost in amyloid precursor protein knock-in (APPKI) mouse model of AD. We demonstrated that loss of mushroom spines occurs as a result of accumulation of extracellular ß-amyloid 42 in APPKI culture media. Our results indicate that extracellular Aß42 acts by overactivating mGluR5 receptor in APPKI neurons, leading to elevated Ca(2+) levels in endoplasmic reticulum, compensatory downregulation of STIM2 expression, impaired synaptic nSOC, and reduced CaMKII activity. Pharmacological inhibition of mGluR5 or overexpression of STIM2 rescued synaptic nSOC and prevented mushroom spine loss in APPKI hippocampal neurons. Our results indicate that downregulation of synaptic STIM2-nSOC-CaMKII pathway causes loss of mushroom synaptic spines in both presenilin and APPKI mouse models of AD. We propose that modulators/activators of this pathway may have a potential therapeutic value for treatment of memory loss in AD. Significance statement: A direct connection between amyloid-induced synaptic mushroom spine loss and neuronal store-operated calcium entry pathway is shown. These results provide strong support for the calcium hypothesis of neurodegeneration and further validate the synaptic store-operated calcium entry pathway as a potential therapeutic target for Alzheimer's disease.

STIM2 protects hippocampal mushroom spines from amyloid synaptotoxicity.

BACKGROUND: Alzheimer disease (AD) is a disease of lost memories. Mushroom postsynaptic spines play a key role in memory storage, and loss of mushroom spines has been proposed to be linked to memory loss in AD. Generation of amyloidogenic peptides and accumulation of amyloid plaques is one of the pathological hallmarks of AD. It is important to evaluate effects of amyloid on stability of mushroom spines. RESULTS: In this study we used in vitro and in vivo models of amyloid synaptotoxicity to investigate effects of amyloid peptides on hippocampal mushroom spines. We discovered that application of Aß42 oligomers to hippocampal cultures or injection of Aß42 oligomers directly into hippocampal region resulted in reduction of mushroom spines and activity of synaptic calcium-calmodulin-dependent kinase II (CaMKII). We further discovered that expression of STIM2 protein rescued CaMKII activity and protected mushroom spines from amyloid toxicity in vitro and in vivo. CONCLUSIONS: Obtained results suggest that downregulation of STIM2-dependent stability of mushroom spines and reduction in activity of synaptic CaMKII is a mechanism of hippocampal synaptic loss in AD model of amyloid synaptotoxicity and that modulators/activators of this pathway may have a potential therapeutic value for treatment of AD.

Calcium signaling, excitability, and synaptic plasticity defects in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) and aging result in impaired ability to store memories, but the cellular mechanisms responsible for these defects are poorly understood. Presenilin 1 (PS1) mutations are responsible for many early-onset familial AD (FAD) cases. The phenomenon of hippocampal long-term potentiation (LTP) is widely used in studies of memory formation and storage. Recent data revealed long-term LTP maintenance (L-LTP) is impaired in PS1-M146V knock-in (KI) FAD mice. To understand the basis for this phenomenon, in the present study we analyzed structural synaptic plasticity in hippocampal cultures from wild type (WT) and KI mice. We discovered that exposure to picrotoxin induces formation of mushroom spines in both WT and KI cultures, but the maintenance of mushroom spines is impaired in KI neurons. This maintenance defect can be explained by an abnormal firing pattern during the consolidation phase of structural plasticity in KI neurons. Reduced frequency of neuronal firing in KI neurons is caused by enhanced calcium-induced calcium release (CICR), enhanced activity of calcium-activated potassium channels, and increased afterhyperpolarization. As a result, "consolidation" pattern of neuronal activity converted to "depotentiation" pattern of neuronal activity in KI neurons. Consistent with this model, we demonstrated that pharmacological inhibitors of CICR (dantrolene), of calcium-activated potassium channels (apamin), and of calcium-dependent phosphatase calcineurin (FK506) are able to rescue structural plasticity defects in KI neurons. Furthermore, we demonstrate that incubation with dantrolene or apamin also rescued L-LTP defects in KI hippocampal slices, suggesting a role for a similar mechanism. This proposed mechanism may be responsible for memory defects in AD but also for age-related memory decline.