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| Evaluation axonopathy during early stage of 5xFAD/GFP mice. (A) Diagram of the whole-brain imaging procedure. (B) Continuous coronal sections taken at 800 µm intervals from a Thy1-GFP-M mice. The thickness of the projection was 20 µm. Scale bar: 1 mm. (C) 3D-reconstructed image of GFP-positive neurons in the whole brain of a Thy1-GFP-M mice. A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral. Scale bar: 1 mm. (D) Schematic diagram illustrating that the neurons were labeled by crossing the 5xFAD mice with the Thy1-GFP-M mice. (E) A coronal brain section revealed that there was no axonopathy from 16-week-old Thy1-GFP-M mice. The thickness of the projection was 20 µm. Scale bar: 1 mm. (F,G) The magnifications of the primary motor area and LS in E (white squares), respectively. Scale bar: 50 µm. (H) A coronal brain section form 16-week-old 5xFAD/GFP mice. The thickness of the projection was 20 µm. Scale bar: 1 mm. (I,J) The magnifications of the primary motor area and LS in H (white squares), respectively. The GFP-positive axonopathic swellings were highlighted with white arrows Scale bar: 50 µm. (K) 3D image of GFP-positive neurons and fibers in the somatosensory areas from 16-week-old 5xFAD/GFP mice. The axonopathic swellings were highlighted with white boxes. Scale bar: 100 µm. (L,M) The three-dimensional image of axons (top) and the surface render of axons showed three-dimensional morphology of axons (bottom) in a 5xFAD/GFP mice. The axonopathy was highlighted with white arrow. Heat-map plots showed the size of axonopathy. Red = bigger size, blue = smaller size. Scale bars: 10 µm both in (H,I).
Source publication
Axonopathy is a pathological feature observed in both Alzheimer's disease (AD) patients and animal models. However, identifying the temporal and regional progression of axonopathy during AD development remains elusive. Using the fluorescence micro-optical sectioning tomography system, we acquired whole-brain datasets in the early stage of 5xFAD/Thy...
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... locate the neuronal soma and identify the individual axon throughout the entire brain without losing crucial information, we generated whole-brain three-dimensional datasets of GFPpositive neurons in 16-week-old Thy1-GFP-M mice by fMOST at a voxel resolution of 0.32 × 0.32 × 2 µm 3 ( Figures 1A,B). Through these continuous three-dimensional datasets, we found that the vast majority of GFP-positive neurons were located in the deep layer of the cortex, hippocampus, and amygdala ( Figure 1C), which is consistent with previous studies ( Feng et al., 2000;Porrero et al., 2010). ...
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... locate the neuronal soma and identify the individual axon throughout the entire brain without losing crucial information, we generated whole-brain three-dimensional datasets of GFPpositive neurons in 16-week-old Thy1-GFP-M mice by fMOST at a voxel resolution of 0.32 × 0.32 × 2 µm 3 ( Figures 1A,B). Through these continuous three-dimensional datasets, we found that the vast majority of GFP-positive neurons were located in the deep layer of the cortex, hippocampus, and amygdala ( Figure 1C), which is consistent with previous studies ( Feng et al., 2000;Porrero et al., 2010). ...
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... evaluate axonal pathological changes in AD, we crossed 5xFAD with Thy1-GFP-M mice ( Figure 1D). Thus, we could analyze axonal morphological changes occurring during aging in the mouse model by using the fMOST system. ...
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... on the whole-brain three-dimensional datasets acquired via the fMOST system, we compared the axonal morphological differences between 5xFAD/GFP mice and Thy1-GFP-M mice. At the same age, compared to the Thy1-GFP-M mice (Figures 1E-G), the 5xFAD/GFP mice exhibited numerous axons with swellings or spheroids in cortical and subcortical regions (Figure 1H), such as the primary motor area (Figure 1I) and the lateral septal nucleus (LS) (Figure 1J). In previous reports, this was observed at later time points (Buskila et al., 2013;Sadleir et al., 2016). ...
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... on the whole-brain three-dimensional datasets acquired via the fMOST system, we compared the axonal morphological differences between 5xFAD/GFP mice and Thy1-GFP-M mice. At the same age, compared to the Thy1-GFP-M mice (Figures 1E-G), the 5xFAD/GFP mice exhibited numerous axons with swellings or spheroids in cortical and subcortical regions (Figure 1H), such as the primary motor area (Figure 1I) and the lateral septal nucleus (LS) (Figure 1J). In previous reports, this was observed at later time points (Buskila et al., 2013;Sadleir et al., 2016). ...
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... on the whole-brain three-dimensional datasets acquired via the fMOST system, we compared the axonal morphological differences between 5xFAD/GFP mice and Thy1-GFP-M mice. At the same age, compared to the Thy1-GFP-M mice (Figures 1E-G), the 5xFAD/GFP mice exhibited numerous axons with swellings or spheroids in cortical and subcortical regions (Figure 1H), such as the primary motor area (Figure 1I) and the lateral septal nucleus (LS) (Figure 1J). In previous reports, this was observed at later time points (Buskila et al., 2013;Sadleir et al., 2016). ...
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... on the whole-brain three-dimensional datasets acquired via the fMOST system, we compared the axonal morphological differences between 5xFAD/GFP mice and Thy1-GFP-M mice. At the same age, compared to the Thy1-GFP-M mice (Figures 1E-G), the 5xFAD/GFP mice exhibited numerous axons with swellings or spheroids in cortical and subcortical regions (Figure 1H), such as the primary motor area (Figure 1I) and the lateral septal nucleus (LS) (Figure 1J). In previous reports, this was observed at later time points (Buskila et al., 2013;Sadleir et al., 2016). ...
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... previous reports, this was observed at later time points (Buskila et al., 2013;Sadleir et al., 2016). To further characterize these pathological changes, we reconstructed the spheroids on the axons of GFPpositive neurons in the somatosensory cortex (Figures 1K-M). The individual axon had multiple pathological sites ( Figure 1L). ...
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... further characterize these pathological changes, we reconstructed the spheroids on the axons of GFPpositive neurons in the somatosensory cortex (Figures 1K-M). The individual axon had multiple pathological sites ( Figure 1L). Discontinuity of the axon was visible around the clustered swellings ( Figure 1M), suggesting axonal disruption as reported in previous studies (Tsai et al., 2004;Adalbert et al., 2009). ...
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... individual axon had multiple pathological sites ( Figure 1L). Discontinuity of the axon was visible around the clustered swellings ( Figure 1M), suggesting axonal disruption as reported in previous studies (Tsai et al., 2004;Adalbert et al., 2009). Our results indicated that axons were severely damaged in 16-weekold 5xFAD/GFP mice. ...
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... study the regional burden and temporal progression of axonopathy more comprehensively at a high spatial resolution, we performed whole-brain imaging from 8-week-old and 16-week-old 5xFAD/GFP mice. As expected, the axonopathy appeared in specific regions during 8 to 16 weeks old, including the isocortex, olfactory area, hippocampal formation, cortical subplate, striatum, thalamus, hypothalamus and fiber tracts (Figure 2A and Supplementary Figure S1). Interestingly, we found that the number of axonal swellings in many brain regions increased, while it seemed to decrease significantly in the MM from 8 to 16 weeks old (Figure 2A). ...
Citations
... Increasing evidence suggests that the swelling of axons and apical dendrites is associated with intraneuronal Aβ accumulation in their cell bodies [91] and this can be a prerequisite for accelerated tau fibrillization. A previous study on axonopathy using whole-brain 3D profiling revealed numerous axonal spheroids in the cortical and subcortical regions of the brains of 5xFAD/ Thy1-GFP mice [92]. Herein, we demonstrated that 3D optical imaging using iACT efficiently visualized the significant loss of mesolimbic DA projections. ...
Optical brain clearing combined with immunolabeling is valuable for analyzing molecular tissue structures, including complex synaptic connectivity. However, the presence of aberrant lipid deposition due to aging and brain disorders poses a challenge for achieving antibody penetration throughout the entire brain volume. Herein, we present an efficient brain-wide immunolabeling method, the immuno-active clearing technique (iACT). The treatment of brain tissues with a zwitterionic detergent, specifically SB3-12, significantly enhanced tissue permeability by effectively mitigating lipid barriers. Notably, Quadrol treatment further refines the methodology by effectively eliminating residual detergents from cleared brain tissues, subsequently amplifying volumetric fluorescence signals. Employing iACT, we uncover disrupted axonal projections within the mesolimbic dopaminergic (DA) circuits in 5xFAD mice. Subsequent characterization of DA neural circuits in 5xFAD mice revealed proximal axonal swelling and misrouting of distal axonal compartments in proximity to amyloid-beta plaques. Importantly, these structural anomalies in DA axons correlate with a marked reduction in DA release within the nucleus accumbens. Collectively, our findings highlight the efficacy of optical volumetric imaging with iACT in resolving intricate structural alterations in deep brain neural circuits. Furthermore, we unveil the compromised integrity of DA pathways, contributing to the underlying neuropathology of Alzheimer’s disease. The iACT technique thus holds significant promise as a valuable asset for advancing our understanding of complex neurodegenerative disorders and may pave the way for targeted therapeutic interventions.
Graphical Abstract
The axonal projection of DA neurons in the septum and the NAc showed dystrophic phenotypes such as growth cone-like enlargement of the axonal terminus and aggregated neurites. Brain-wide imaging of structural defects in the neural circuits was facilitated with brain clearing and antibody penetration assisted with SB3-12 and Quadrol pre-treatment. The whole volumetric imaging process could be completed in a week with the robust iACT method. Created with https://www.biorender.com/ .
... 6 A precise segmentation of 3D neuron structures is vital to probe impaired brain functions of AD animal models and to determine early treatment strategies. 7 We might gain a better understanding of such disorders if we could find specific neuronal morphology in disease models. 8 Through the accurate signals of neuron morphology, we can systematically classify brain cells. ...
Significance:
Robust segmentations of neurons greatly improve neuronal population reconstruction, which could support further study of neuron morphology for brain research.
Aim:
Precise segmentation of 3D neuron structures from optical microscopy (OM) images is crucial to probe neural circuits and brain functions. However, the high noise and low contrast of images make neuron segmentation challenging. Convolutional neural networks (CNNs) can provide feasible solutions for the task but they require large manual labels for training. Labor-intensive labeling is highly expensive and heavily limits the algorithm generalization.
Approach:
We devise a weakly supervised learning framework Docker-based deep network plus (DDeep3M+) for neuron segmentation without any manual labeling. A Hessian analysis based adaptive enhancement filter is employed to generate pseudo-labels for segmenting neuron images. The automated segmentation labels are input for training a DDeep3M to extract neuronal features. We mine more undetected weak neurites from the probability map based on neuronal structures, thereby modifying the pseudo-labels. We iteratively refine the pseudo-labels and retrain the DDeep3M model with the pseudo-labels to obtain a final segmentation result.
Results:
The proposed method achieves promising results with the F1 score of 0.973, which is close to that of the CNN model with manual labels and superior to several segmentation algorithms.
Conclusions:
We propose an accurate weakly supervised neuron segmentation method. The high precision results achieved on 3D OM datasets demonstrate the superior generalization of our DDeep3M+.
... 10 In addition, staining and observation of pathological features such as amyloid-b plaque labeled by immunostaining or fluorescein probe contribute to understanding the pathogenesis of Alzheimer's disease (AD). 11 Therefore, to better understand the interaction of the various components of the brain, we should acquire the fluorescent signals of multiple fine structures with sub-micron resolution. ...
Resin embedding combined with ultra-thin sectioning has been widely used in microscopic and electron imaging to acquire precise structural information of biological tissues. However, the existing embedding method was detrimental to quenchable fluorescent signals of precise structures and pH-insensitive fluorescent dyes. Here, we developed a low-temperature chemical polymerization method named HM20-T to maintain weak signals of various precise structures and to decrease background fluorescence. The fluorescence preservation ratio of green fluorescent protein (GFP) tagged presynaptic elements and tdTomato labeled axons doubled. The HM20-T method was suitable for a variety of fluorescent dyes, such as DyLight 488 conjugated Lycopersicon esculentum lectin. Moreover, the brains also retained immunoreactivity after embedding. In summary, the HM20-T method was suitable for the characterization of multi-color labeled precise structures, which would contribute to the acquisition of complete morphology of various biological tissues and to the investigation of composition and circuit connection in the whole brain.
... Whereas in later stages of AD the affected cells appear to be more generic. These approaches will likely need to be complemented with other neuroanatomical imaging modalities [37,38], which will reveal cell type-specific circuit structural changes early in AD progression. ...
The study of Alzheimer’s Disease (AD) has traditionally focused on neuropathological mechanisms that has guided therapies that attenuate neuropathological features. A new direction is emerging in AD research that focuses on the progressive loss of cognitive function due to disrupted neural circuit mechanisms. Evidence from humans and animal models of AD show that dysregulated circuits initiate a cascade of pathological events that culminate in functional loss of learning, memory, and other aspects of cognition. Recent progress in single-cell, spatial, and circuit omics informs this circuit-focused approach by determining the identities, locations, and circuitry of the specific cells affected by AD. Recently developed neuroscience tools allow for precise access to cell type-specific circuitry so that their functional roles in AD-related cognitive deficits and disease progression can be tested. An integrated systems-level understanding of AD-associated neural circuit mechanisms requires new multimodal and multi-scale interrogations that longitudinally measure and/or manipulate the ensemble properties of specific molecularly-defined neuron populations first susceptible to AD. These newly developed technological and conceptual advances present new opportunities for studying and treating circuits vulnerable in AD and represent the beginning of a new era for circuit-based AD research.
... Authors showed that among GFP-labeled axons, GFP-labeled axonopathy underwent early alterations in the lateral septal nucleus, subiculum, and mammillary nucleus in the 5xFAD/GFP mouse model. It is important to point out that authors have not been evaluating the dendritic spine morphology in this mouse model [31]. ...
Cognitive impairments are closely related to synaptic loss in Alzheimer’s disease (AD). Functional changes in synaptic contacts are reflected in dendritic spine morphology. Visualization of neurons for morphological studies in vivo is complicated by the fixed brain slice staining or expensive adeno-associated virus injections. We created a transgenic 5xFAD-M line of mice with AD-associated mutations and expressed GFP protein in single neurons of the brain. This mouse model of AD is a useful tool for the simplified visualization of the hippocampal neurons’ morphology in vivo without additional staining manipulations. The progressive elimination of mushroom spines was demonstrated in 5xFAD-M mice between 4 and 5 months of age. Five-month-old 5xFAD-M male and female mice showed change both in the total density and the mushroom spines number compared to sex-matched control. We conclude 5xFAD-M mice can be a useful AD model for studying the mechanisms of synaptic pathology under neurodegenerative conditions and evaluating the effects of potential therapeutic agents on spine morphology as crucial aspect of memory loss in AD.
... The 5×FAD mouse line expresses the human APP and PSEN1 transgenes 35,36 . CNTNAP3KO mouse was a gift from Zilong Qiu Lab 37 . ...
Dissection of the anatomical information at the single-cell level is crucial for understanding the organization rule and pathological mechanism of biological tissues. Mapping the whole organ in numerous groups with multiple conditions brings the challenges in imaging and analysis. Here, we describe an approach, named array fluorescent micro-optical sectioning tomography (array-fMOST), to identify the three-dimensional information at single-cell resolution from multi-samples. The pipeline contains array embedding, large-scale imaging, post-imaging staining and data analysis, which could image over 24 mouse brains simultaneously and collect the slices for further analysis. With transgenic mice, we acquired the distribution information of neuropeptide somatostatin neurons during natural aging and compared the changes in the microenvironments by multi-component labeling of serial sections with precise co-registration of serial datasets quantitatively. With viral labeling, we also analyzed the input circuits of the medial prefrontal cortex in the whole brain of Alzheimer’s disease and autism model mice. This pipeline is highly scalable to be applied to anatomical alterations screening and identification. It provides new opportunities for combining multi-sample whole-organ imaging and molecular phenotypes identification analysis together. Such integrated high-dimensional information acquisition method may accelerate our understanding of pathogenesis and progression of disease in situ at multiple levels.
... Thus, it is very likely that different PV + neurons innervated the HPF and hypothalamus. Together with the results of our previous study of axonopathy and Aβ plaques in the whole brain (35,42), a pathway-specific manner of neuron degeneration was observed, in which specific neuron types were particularly vulnerable to AD progression. The results of the present study showed that the density of Aβ plaques in the MM was higher, while the number of PV + synapses in the MM showed a greater decrease than that in CA3 (Fig. 4 C and F), which was consistent with the original assumptions that synapses degenerate, particularly near amyloid plaques (43). ...
Through synaptic connections, long-range circuits transmit information among neurons and connect different brain regions to form functional motifs and execute specific functions. Tracing the synaptic distribution of specific neurons requires submicron-level resolution information. However, it is a great challenge to map the synaptic terminals completely because these fine structures span multiple regions, even in the whole brain. Here, we develop a pipeline including viral tracing, sample embedding, fluorescent micro-optical sectional tomography, and big data processing. We mapped the whole-brain distribution and architecture of long projections of the parvalbumin neurons in the basal forebrain at the synaptic level. These neurons send massive projections to multiple downstream regions with subregional preference. With three-dimensional reconstruction in the targeted areas, we found that synaptic degeneration was inconsistent with the accumulation of amyloid-β plaques but was preferred in memory-related circuits, such as hippocampal formation and thalamus, but not in most hypothalamic nuclei in 8-month-old mice with five familial Alzheimer’s disease mutations. Our pipeline provides a platform for generating a whole-brain atlas of cell-type-specific synaptic terminals in the physiological and pathological brain, which can provide an important resource for the study of the organizational logic of specific neural circuits and the circuitry changes in pathological conditions.
... The previous studies employing brain-wide imaging techniques have realized the brain-wide visualization of Aβ plaques (Liebmann et al., 2016;Long et al., 2019;Whitesell et al., 2019). Besides, whole-brain imaging of the single surrounding structure such as blood vessels, cells, and neural circuits associated with Aβ plaques was also achieved easily (Meyer et al., 2008;Liebmann et al., 2016;Zhang et al., 2020). Nevertheless, a few studies have systematically correlated the other multiple surrounding structures morphology with their spatial relation to Aβ plaques, especially at the submicron level in the intact brain. ...
Simultaneously visualizing Amyloid-β (Aβ) plaque with its surrounding brain structures at the subcellular level in the intact brain is essential for understanding the complex pathology of Alzheimer's disease, but is still rarely achieved due to the technical limitations. Combining the micro-optical sectioning tomography (MOST) system, whole-brain Nissl staining, and customized image processing workflow, we generated a whole-brain panorama of Alzheimer's disease mice without specific labeling. The workflow employed the steps that include virtual channel splitting, feature enhancement, iso-surface rendering, direct volume rendering, and feature fusion to extract and reconstruct the different signals with distinct gray values and morphologies. Taking advantage of this workflow, we found that the denser-distribution areas of Aβ plaques appeared with relatively more somata and smaller vessels, but show a dissimilar distributing pattern with nerve tracts. In addition, the entorhinal cortex and adjacent subiculum regions present the highest density and biggest diameter of plaques. The neuronal processes in the vicinity of these Aβ plaques showed significant structural alternation such as bending or abrupt branch ending. The capillaries inside or adjacent to the plaques were observed with abundant distorted micro-vessels and abrupt ending. Depicting Aβ plaques, somata, nerve processes and tracts, and blood vessels simultaneously, this panorama enables us for the first time, to analyze how the Aβ plaques interact with capillaries, somata, and processes at a submicron resolution of 3D whole-brain scale, which reveals potential pathological effects of Aβ plaques from a new cross-scale view. Our approach opens a door to routine systematic studies of complex interactions among brain components in mouse models of Alzheimer's disease.
Background
Bioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer’s, Parkinson’s, and Huntington’s disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function.
Methods
We generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of techniques, including genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos.
Results
We provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide “on-board” ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD ⁺ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons.
Conclusion
NMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.
