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Neurodegenerative diseases are complex and progressive, posing challenges to their study and understanding. Recent advances in microscopy imaging technologies have enabled the exploration of neurons in three spatial dimensions (3D) over time (4D). When applied to 3D cultures, tissues, or animals, these technologies can provide valuable insights into the dynamic and spatial nature of neurodegenerative diseases. This review focuses on the use of imaging techniques and neurodegenerative disease models to study neurodegeneration in 4D. Imaging techniques such as confocal microscopy, two-photon microscopy, miniscope imaging, light sheet microscopy, and robotic microscopy offer powerful tools to visualize and analyze neuronal changes over time in 3D tissue. Application of these technologies to in vitro models of neurodegeneration such as mouse organotypic culture systems and human organoid models provide versatile platforms to study neurodegeneration in a physiologically relevant context. Additionally, use of 4D imaging in vivo, including in mouse and zebrafish models of neurodegenerative diseases, allows for the investigation of early dysfunction and behavioral changes associated with neurodegeneration. We propose that these studies have the power to overcome the limitations of two-dimensional monolayer neuronal cultures and pave the way for improved understanding of the dynamics of neurodegenerative diseases and the development of effective therapeutic strategies., Competing Interests: Conflict of interest S. F. is the inventor of Robotic Microscopy Systems, US Patent 7,139,415 and Automated Robotic Microscopy Systems, US Patent Application 14/737,325, both assigned to the J. David Gladstone Institutes. A provisional US and EPO patent for the GEDI biosensor (inventors J. W. L. and S. F.) assigned to the J. David Gladstone Institutes has been placed GL2016 to 815, May 2019. S. F. and J. W. L. are co-founders of Operant Biopharma. The other author declares that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Excess gene dosage from chromosome 21 (chr21) causes Down syndrome (DS), spanning developmental and acute phenotypes in terminal cell types. Which phenotypes remain amenable to intervention after development is unknown. To address this question in a model of DS neurogenesis, we derived trisomy 21 (T21) human induced pluripotent stem cells (iPSCs) alongside, otherwise, isogenic euploid controls from mosaic DS fibroblasts and equipped one chr21 copy with an inducible XIST transgene. Monoallelic chr21 silencing by XIST is near-complete and irreversible in iPSCs. Differential expression reveals that T21 neural lineages and iPSCs share suppressed translation and mitochondrial pathways and activate cellular stress responses. When XIST is induced before the neural progenitor stage, T21 dosage correction suppresses a pronounced skew toward astrogenesis in neural differentiation. Because our transgene remains inducible in postmitotic T21 neurons and astrocytes, we demonstrate that XIST efficiently represses genes even after terminal differentiation, which will empower exploration of cell type-specific T21 phenotypes that remain responsive to chr21 dosage.

Neurotoxicity can be detected in live microscopy by morphological changes such as retraction of neurites, fragmentation, blebbing of the neuronal soma and ultimately the disappearance of fluorescently labeled neurons. However, quantification of these features is often difficult, low-throughput, and imprecise due to the overreliance on human curation. Recently, we showed that convolutional neural network (CNN) models can outperform human curators in the assessment of neuronal death from images of fluorescently labeled neurons, suggesting that there is information within the images that indicates toxicity but that is not apparent to the human eye. In particular, the CNN's decision strategy indicated that information within the nuclear region was essential for its superhuman performance. Here, we systematically tested this prediction by comparing images of fluorescent neuronal morphology from nuclear-localized fluorescent protein to those from freely diffused fluorescent protein for classifying neuronal death. We found that biomarker-optimized (BO-) CNNs could learn to classify neuronal death from fluorescent protein-localized nuclear morphology (mApple-NLS-CNN) alone, with super-human accuracy. Furthermore, leveraging methods from explainable artificial intelligence, we identified novel features within the nuclear-localized fluorescent protein signal that were indicative of neuronal death. Our findings suggest that the use of a nuclear morphology marker in live imaging combined with computational models such mApple-NLS-CNN can provide an optimal readout of neuronal death, a common result of neurotoxicity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Linsley, Linsley, Lamstein and Finkbeiner.)

Cellular events underlying neurodegenerative disease may be captured by longitudinal live microscopy of neurons. While the advent of robot-assisted microscopy has helped scale such efforts to high-throughput regimes with the statistical power to detect transient events, time-intensive human annotation is required. We addressed this fundamental limitation with biomarker-optimized convolutional neural networks (BO-CNNs): interpretable computer vision models trained directly on biosensor activity. We demonstrate the ability of BO-CNNs to detect cell death, which is typically measured by trained annotators. BO-CNNs detected cell death with superhuman accuracy and speed by learning to identify subcellular morphology associated with cell vitality, despite receiving no explicit supervision to rely on these features. These models also revealed an intranuclear morphology signal that is difficult to spot by eye and had not previously been linked to cell death, but that reliably indicates death. BO-CNNs are broadly useful for analyzing live microscopy and essential for interpreting high-throughput experiments.

Cell death is a critical process that occurs normally in health and disease. However, its study is limited due to available technologies that only detect very late stages in the process or specific death mechanisms. Here, we report the development of a family of fluorescent biosensors called genetically encoded death indicators (GEDIs). GEDIs specifically detect an intracellular Ca 2+ level that cells achieve early in the cell death process and that marks a stage at which cells are irreversibly committed to die. The time-resolved nature of a GEDI delineates a binary demarcation of cell life and death in real time, reformulating the definition of cell death. We demonstrate that GEDIs acutely and accurately report death of rodent and human neurons in vitro, and show that GEDIs enable an automated imaging platform for single cell detection of neuronal death in vivo in zebrafish larvae. With a quantitative pseudo-ratiometric signal, GEDIs facilitate high-throughput analysis of cell death in time-lapse imaging analysis, providing the necessary resolution and scale to identify early factors leading to cell death in studies of neurodegeneration., (© 2021. The Author(s).)

Neuropeptides are important for regulating numerous neural functions and behaviors. Release of neuropeptides requires long-lasting, high levels of cytosolic Ca 2+ However, the molecular regulation of neuropeptide release remains to be clarified. Recently, Stac3 was identified as a key regulator of L-type Ca 2+ channels (CaChs) and excitation-contraction coupling in vertebrate skeletal muscles. There is a small family of stac genes in vertebrates with other members expressed by subsets of neurons in the central nervous system. The function of neural Stac proteins, however, is poorly understood. Drosophila melanogaster contain a single stac gene, Dstac , which is expressed by muscles and a subset of neurons, including neuropeptide-expressing motor neurons. Here, genetic manipulations, coupled with immunolabeling, Ca 2+ imaging, electrophysiology, and behavioral analysis, revealed that Dstac regulates L-type CaChs (Dmca1D) in Drosophila motor neurons and this, in turn, controls the release of neuropeptides., Competing Interests: The authors declare no competing interest.

Stac3 regulates excitation-contraction coupling (EC coupling) in vertebrate skeletal muscles by regulating the L-type voltage-gated calcium channel (Ca v channel). Recently a stac -like gene, Dstac , was identified in Drosophila and found to be expressed by both a subset of neurons and muscles. Here, we show that Dstac and Dmca1D, the Drosophila L-type Ca v channel, are necessary for normal locomotion by larvae. Immunolabeling with specific antibodies against Dstac and Dmca1D found that Dstac and Dmca1D are expressed by larval body-wall muscles. Furthermore, Ca 2+ imaging of muscles of Dstac and Dmca1D deficient larvae found that Dstac and Dmca1D are required for excitation-contraction coupling. Finally, Dstac appears to be required for normal expression levels of Dmca1D in body-wall muscles. These results suggest that Dstac regulates Dmca1D during EC coupling and thus muscle contraction., (Copyright © 2020 Hsu, Linsley, Reid, Hume, Leflein and Kuwada.)

Stress granules (SGs) form during cellular stress and are implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). To yield insights into the role of SGs in pathophysiology, we performed a high-content screen to identify small molecules that alter SG properties in proliferative cells and human iPSC-derived motor neurons (iPS-MNs). One major class of active molecules contained extended planar aromatic moieties, suggesting a potential to intercalate in nucleic acids. Accordingly, we show that several hit compounds can prevent the RNA-dependent recruitment of the ALS-associated RNA-binding proteins (RBPs) TDP-43, FUS, and HNRNPA2B1 into SGs. We further demonstrate that transient SG formation contributes to persistent accumulation of TDP-43 into cytoplasmic puncta and that our hit compounds can reduce this accumulation in iPS-MNs from ALS patients. We propose that compounds with planar moieties represent a promising starting point to develop small-molecule therapeutics for treating ALS/FTD., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction : Neurodegenerative diseases affect millions of people worldwide. Neurodegeneration is gradual over time, characterized by neuronal death that causes deterioration of cognitive or motor functions, ultimately leading to the patient's death. Currently, there are no treatments that effectively slow the progression of any neurodegenerative disease, but improved microscopy assays and models for neurodegeneration could lead the way to the discovery of disease-modifying therapeutics. Areas covered : Herein, the authors describe cell-based assays used to discover drugs with the potential to slow neurodegeneration, and their associated disease models. They focus on microscopy technologies that can be adapted to a high-throughput screening format that both detect cell death and monitor early signs of neurodegeneration and functional changes to identify drugs that the block early stages of neurodegeneration. Expert opinion : Many different phenotypes have been used in screens for the development of therapeutics towards neurodegenerative disease. The context of each phenotype in relation to neurodegeneration must be established to identify therapeutics likely to successfully target and treat disease. The use of improved models of neurodegeneration, statistical analyses, computational models, and improved markers of neuronal death will help in this pursuit and lead to better screening methods to identify therapeutic compounds against neurodegenerative disease.

Current approaches for dynamic profiling of single cells rely on dissociated cultures, which lack important biological features existing in tissues. Organotypic slice cultures preserve aspects of structural and synaptic organisation within the brain and are amenable to microscopy, but established techniques are not well adapted for high throughput or longitudinal single cell analysis. Here we developed a custom-built, automated confocal imaging platform, with improved organotypic slice culture and maintenance. The approach enables fully automated image acquisition and four-dimensional tracking of morphological changes within individual cells in organotypic cultures from rodent and human primary tissues for at least 3 weeks. To validate this system, we analysed neurons expressing a disease-associated version of huntingtin (HTT586Q138-EGFP), and observed that they displayed hallmarks of Huntington's disease and died sooner than controls. By facilitating longitudinal single-cell analyses of neuronal physiology, our system bridges scales necessary to attain statistical power to detect developmental and disease phenotypes., Competing Interests: S.M.F. is the inventor of Robotic Microscopy Systems, U.S. Patent 7,139,415 and Automated Robotic Microscopy Systems, U.S. Patent Application 14/737,325, both assigned to The J. David Gladstone Institutes. The remaining authors declare no competing interests.

The genetic, molecular and neuronal mechanism underlying circadian activity rhythms is well characterized in the brain of Drosophila. The small ventrolateral neurons (s-LN V s) and pigment dispersing factor (PDF) expressed by them are especially important for regulating circadian locomotion. Here we describe a novel gene, Dstac, which is similar to the stac genes found in vertebrates that encode adaptor proteins, which bind and regulate L-type voltage-gated Ca 2+ channels (CaChs). We show that Dstac is coexpressed with PDF by the s-LN V s and regulates circadian activity. Furthermore, the L-type CaCh, Dmca1D, appears to be expressed by the s-LN V s. Since vertebrate Stac3 regulates an L-type CaCh we hypothesize that Dstac regulates Dmca1D in s-LN V s and circadian activity.

Contraction of skeletal muscle is initiated by excitation-contraction (EC) coupling during which membrane voltage is transduced to intracellular Ca 2+ release. EC coupling requires L-type voltage gated Ca 2 + channels (the dihydropyridine receptor or DHPR) located at triads, which are junctions between the transverse (T) tubule and sarcoplasmic reticulum (SR) membranes, that sense membrane depolarization in the T tubule membrane. Reduced EC coupling is associated with ageing, and disruptions of EC coupling result in congenital myopathies for which there are few therapies. The precise localization of DHPRs to triads is critical for EC coupling, yet trafficking of the DHPR to triads is not well understood. Using dynamic imaging of zebrafish muscle fibers, we find that DHPR is transported along the longitudinal SR in a microtubule-independent mechanism. Furthermore, transport of DHPR in the SR membrane is differentially affected in null mutants of Stac3 or DHPRβ, two essential components of EC coupling. These findings reveal previously unappreciated features of DHPR motility within the SR prior to assembly at triads., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Skeletal muscle contractions are initiated by an increase in Ca 2+ released during excitation-contraction (EC) coupling, and defects in EC coupling are associated with human myopathies. EC coupling requires communication between voltage-sensing dihydropyridine receptors (DHPRs) in transverse tubule membrane and Ca 2+ release channel ryanodine receptor 1 (RyR1) in the sarcoplasmic reticulum (SR). Stac3 protein (SH3 and cysteine-rich domain 3) is an essential component of the EC coupling apparatus and a mutation in human STAC3 causes the debilitating Native American myopathy (NAM), but the nature of how Stac3 acts on the DHPR and/or RyR1 is unknown. Using electron microscopy, electrophysiology, and dynamic imaging of zebrafish muscle fibers, we find significantly reduced DHPR levels, functionality, and stability in stac3 mutants. Furthermore, stac3 NAM myofibers exhibited increased caffeine-induced Ca 2+ release across a wide range of concentrations in the absence of altered caffeine sensitivity as well as increased Ca 2+ in internal stores, which is consistent with increased SR luminal Ca 2+ These findings define critical roles for Stac3 in EC coupling and human disease., Competing Interests: The authors declare no conflict of interest.

Excitation-contraction coupling, the process that regulates contractions by skeletal muscles, transduces changes in membrane voltage by activating release of Ca(2+) from internal stores to initiate muscle contraction. Defects in excitation-contraction coupling are associated with muscle diseases. Here we identify Stac3 as a novel component of the excitation-contraction coupling machinery. Using a zebrafish genetic screen, we generate a locomotor mutation that is mapped to stac3. We provide electrophysiological, Ca(2+) imaging, immunocytochemical and biochemical evidence that Stac3 participates in excitation-contraction coupling in muscles. Furthermore, we reveal that a mutation in human STAC3 is the genetic basis of the debilitating Native American myopathy (NAM). Analysis of NAM stac3 in zebrafish shows that the NAM mutation decreases excitation-contraction coupling. These findings enhance our understanding of both excitation-contraction coupling and the pathology of myopathies.