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

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

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

Abstract 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

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

Additional file 3: of STIM2 protects hippocampal mushroom spines from amyloid synaptotoxicity

Supplementary experimental procedure. Atomic force microscopy. Imaging was performed on commercial SPM Solver P47-Pro atomic force microscope with NSG 11 probe (Nt-MDT Co., Zelenograd, Moscow, Russia). Images were taken in air using tapping mode on highly ordered pyrolytic graphite (Nt-MDT Co., Zelenograd, Moscow, Russia). The surface of graphite was carefully rinsed with deionized water and gently dried under a N2 stream. NovaRC1 operating software version 850 was used to acquire the data images, Gwyddion and SPIP software were used to render the data and perform the analyses. The z-height of 9-10 globules from two different areas on the graphite was measured. Particle analysis was performed by choosing a threshold height equivalent to 1/2 the average z-height of the globules. This measures globule diameter at fwhm (full width at half-maximum). To get approximate width of globules geometrical deconvolution model [36] was used, particles were treated as spheres h = w / 2 2 R t $$\mathrm{h}=\raisebox{1ex}{$\sqrt{w/2}$}\!\left/ \!\raisebox{-1ex}{$2Rt$}\right.$$ where h is the real width of the structure, w is the width or diameter observed in the AFM image, and Rt is the tip apex radius (in our case it is around 10 nm). (DOCX 12 kb

Additional file 2: Figure S2. of STIM2 protects hippocampal mushroom spines from amyloid synaptotoxicity

Visualization of hippocampal neurons injected with Alexa-555 labelled Aβ42. Low-magnification image of CA1 hippocampal area from 3.5 months old Thy1-GFP line M mouse is presented. Image is taken six weeks after injection. CA1 neurons expressing GFP protein are shown in green, signals from Alexa-555 labeled Aβ42 are shown in red. The specific dendritic segment where the data on spine density and shape were obtained is marked with white circle. Scale bar corresponds to 20 μm. (PDF 51 kb

Additional file 1: Figure S1. of STIM2 protects hippocampal mushroom spines from amyloid synaptotoxicity

Characterization of oligomeric state of Aβ42, Aβ40 and Aβ42-Alexa555. (A) Atomic force microscopy (performed as described in Additonal file 3) images of Aβ42, Aβ40 and Aβ42-Alexa555 samples after 24 h incubation at 4 °C visualized on graphite. Sizes of field of view for left images are 4×4μm and for right images 1×1μm. Topographic profile for each Aβ sample is presented (measured globule is marked with blue line). All Aβ samples appear primarily as globular structures with following sizes: Aβ42 height 1.56 ± 0.3 nm, diameter at fwhm before deconvolution 75 ± 0.6 nm, diameter at fwhm after deconvolution 9.6 ± 0.1 nm; Aβ40 height 0.9 ± 0.1 nm, diameter at fwhm before deconvolution 43 ± 0.3 nm, diameter at fwhm after deconvolution 7.3 ± 0.1 nm; Aβ42-Alexa555 height 1.46 ± 0.2 nm, diameter at fwhm before deconvolution 68 ± 0.6 nm, diameter at fwhm after deconvolution 8.9 ± 0.1 nm. (B) Supernatant fractions of Aβ42, Aβ40 and Aβ42-Alexa555 preparations were separated on 15 % Acrylamide/Bis SDS gel and analyzed by Western blotting with anti-Aβ 6E10 monolconal antibodies. (PDF 375 kb