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| Morphology of axonal varicosities in injured, diseased or normal brains. (A), Post-mortem APP immunoreactivity within the corpus callosum of a TBI patient displayed multiple varicosities (blue arrows) along individual axons (Tang-Schomer et al., 2012). The swelling without a visible thin linking axon is a potential terminal bulb (red arrow). Scale bar, 30 µm. (B), The transmission electron microscope (TEM) image of an axonal varicosity or swelling filled with neurofibrils (NF) from the post-mortem cerebral cortex of a patient with Alzheimer's presenile dementia (Terry et al., 1964). Scale bar, 1 µm. (C), Ultrastructure of axonal varicosity in a mouse model of AD (Stokin et al., 2005). Degenerative changes in axonal varicosity along the fibers of the nucleus basalis of Meynert in Tg-swAPPPrp (b) but not wild-type mice (a). Scale bar, 1 µm. (D), TEM images of cultured cortical neurons from spastin −/− mice show disorganization of the microtubule (MT) network within axonal varicosities (Fassier et al., 2013). The image in the lower panel is the high magnification of axonal varicosity indicated by a blackarrow in the upper image. The image on the right is the high magnification of boxed regions, showing the tangled and bent aspect of MT filaments within the axonal varicosity. This abnormal appearance of MTs was never observed in wild-type axons, in which MTs were always organized in parallel arrays. White arrows indicate MT. Scale bars: 5 µm (upper left), 0.5 µm (lower left) and 0.25 µm (right). (E), Ultrastructural features of CA3 varicosities (Var) and axons in stratum radiatum of area CA1 in the normal rat brain (Shepherd and Harris, 1998). Longitudinally sectioned axon with two boutons, neither of which had additional PSDs or mitochondria in adjacent images. mito, mitochondria; MSB, multiple-synapse bouton; PSD, postsynaptic density. Scale bar, 1 µm. (F), Eight reconstructed axons from series electron microscopy images including axonal varicosities (Shepherd and Harris, 1998).
Source publication
Axonal varicosities are enlarged, heterogeneous structures along axonal shafts, profoundly affecting axonal conduction and synaptic transmission. They represent a key pathological feature believed to develop via slow accumulation of axonal damage that occurs during irreversible degeneration, for example in mild traumatic brain injury (mTBI), Alzhei...
Contexts in source publication
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... damaged axons can be labeled with various markers. In post-mortem APP (amyloid precursor protein) immunostaining of the corpus callosum, no signal is normally observed in a healthy human subject, whereas staining signals from TBI patients displayed a classic pattern of axonal varicosities-multiple individual varicosities along the length of an individual axon, as well as potential terminal bulbs without narrow axonal shaft linking them ( Figure 1A) (Tang-Schomer et al., 2012). In the past, the development of axonal varicosities was thought very slow and irreversible, preceding the formation of terminal bulbs in broken or degenerated axons. ...
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... is an irreversible, progressive brain disorder showing slow impairment of memory and thinking skills. Ultrastructural studies using electron microscopy showed various axonal pathologies including axonal varicosities filled with neurofibrils in the brain of human patients with AD ( Figure 1B) (Terry et al., 1964). In 4-month-old TgswAPPPrp mice, a mouse model for AD, axonal varicosities that were positive or negative for choline acetyltransferase (ChAT) in the nucleus basalis of Meynert were analyzed by comparing with wild-type mice (Stokin et al., 2005). ...
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... varicosities contained large numbers of organelles and vesicles, and were not myelinated, nor associated with postsynaptic densities. A subset of varicosities displayed characteristics of axonal degeneration, including electron-dense granular axoplasm and a large amount of axoplasmic debris of tubular appearance ( Figure 1C) (Stokin et al., 2005). ...
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... corticospinal tracts (Fink, 2006). As a key feature for progressive axonal degeneration, axonal varicosities developed with disorganized microtubule filaments and hence impaired axonal transport in cortical neurons of SPG4 knockout mice ( Figure 1D) (Fassier et al., 2013). Taken together, since axonal varicosities were considered merely as a subcellular structure preceding axon degeneration, early studies did not make a major effort to distinguish axonal varicosities from terminal bulbs, nor to understand the transition from a varicosity to a terminal bulb. ...
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... axonal varicosities started to form from 5 to 10 min after pulling and some appeared to form retraction bulbs of broken axons (Hemphill et al., 2011). The authors further showed that Rho-kinase inhibitor but not calpain inhibitor significantly reduced axonal varicosity formation and axonal injury in response to either 5 or 10% strain, suggesting an important role of integrin-mediated activation of Rho in the process (Hemphill et al., 2011). ...
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... varicosity formation was also observed from openskull impact TBI models. Using a central fluid percussion injury (FPI) model, the Povlishock group showed that axonal varicosities in the optic nerve formed as early as 5-15 min post injury, and appeared to associate with an accumulation of mitochondria under electron microscopy (Wang et al., 2011). By combining the FPI model and GABAergic interneuron markers, the same group reported that axonal varicosities were developed 3 h post injury in the cortex (Figure 4E) (Vascak et al., 2018). ...
Citations
... Varicosities significantly impair the conduction of a nerve impulse along the axon and its transsynaptic transmission [31]. The appearance of axonal varicosities in neurons is characteristic of various neurological disorders, including Alzheimer's disease, PD, and multiple sclerosis, and apparently reflects an increase in pathological changes in the axon during irreversible neurodegeneration [32]. In our culture with SNCA mutation, many cells contained a large nucleus and a large volume of cytoplasm which was poor in organelles, and this, in our opinion, corresponds to the ultrastructural features of immature neurons (neuronal progenitors). ...
Parkinson’s disease (PD) is the second most common neurodegenerative disease. Some cases of PD may be caused by genetic factors, among which mutations in the LRRK2 and SNCA genes play an important role. To develop effective neuroprotective strategies for PD, it is important to diagnose the disease at the earliest stages of the neurodegenerative process. Therefore, the detection of diagnostic and prognostic markers of Parkinson’s disease (PD) is an urgent medical need. Advances in induced pluripotent stem cell (iPSC) culture technology provide new opportunities for the search for new biomarkers of PD and its modeling in vitro. In our work, we used a new technology for multiplex profiling of gene expression using barcoding on the Nanostring platform to assess the activity of mitochondrial genes on iPSC-derived cultures of dopaminergic neurons obtained from patients with LRRK2- and SNCA-associated genetic forms PD and a healthy donor. Electron microscopy revealed ultrastructural changes in mitochondria in both LRRK2 and SNCA mutant cells, whereas mitochondria in cells from a healthy donor were normal. In a culture with the SNCA gene mutation, the ratio of the area occupied by mitochondria to the total area of the cytoplasm was significantly lower than in the control and in the line with the LRRK2 gene mutation. Transcriptome analysis of 105 mitochondria proteome genes using the Nanostring platform revealed differences between the diseased and normal cells in the activity of genes involved in respiratory complex function, the tricarboxylic acid cycle, ATP production, mitochondria–endoplasmic reticulum interaction, mitophagy, regulation of calcium concentration, and mitochondrial DNA replication.
... Serotonergic varicosities are likely to be fluid structures, as suggested by experimental studies of mice and Drosophila with elevated serotonin levels (Daubert et al., 2010), a rat model of epilepsy (Maia et al., 2019), and analyses of varicosities in other neurotransmitter systems. Specifically, the appearance of varicosities may reflect the current state of the fiber segment, the effects of its local microenvironment, and purely stochastic fluctuations (Hellwig et al., 1994;Hatada et al., 1999;Shepherd et al., 2002;Gu, 2021;Ma et al., 2022). Notably, the dynamics of varicosities on the same fiber are likely to be correlated, necessitating unambiguous discrimination among individual fibers that often cross and intertwine in the same spatial location. ...
The self-organization of the serotonergic matrix, a massive axon meshwork in all vertebrate brains, is driven by the structural and dynamical properties of its constitutive elements. Each of these elements, a single serotonergic axon (fiber), has a unique trajectory and can be supported by a soma that executes one of the many available transcriptional programs. This “individuality” of serotonergic neurons necessitates the development of specialized methods for single-fiber analyses, both at the experimental and theoretical levels. We developed an integrated platform that facilitates experimental isolation of single serotonergic fibers in brain tissue, including regions with high fiber densities, and demonstrated the potential of their quantitative analyses based on stochastic modeling. Single fibers were visualized using two transgenic mouse models, one of which is the first implementation of the Brainbow toolbox in this system. The trajectories of serotonergic fibers were automatically traced in the three spatial dimensions with a novel algorithm, and their properties were captured with a single parameter associated with the directional von Mises-Fisher probability distribution. The system represents an end-to-end workflow that can be imported into various studies, including those investigating serotonergic dysfunction in brain disorders. It also supports new research directions inspired by single-fiber analyses in the serotonergic matrix, including supercomputing simulations and modeling in physics.
... Although the function of dendritic varicosities is still not fully understood, it is known that amacrine dendritic varicosities electrically isolate local input-output neuronal circuits [110]. Axonal varicosities, which are involved in the antidromic propagation of action potentials to the soma in a retrograde manner [111], likely play a role in neuron mechanosensation [112] and protection [113] in the CNS, and contribute to blood flow regulation in the peripheral nervous system [114]. The observed varicosities suggest the activation of neurons. ...
Introduction:
While most animals of the Muridae family are nocturnal, the gerbil displays diurnal activity and provides a useful model for visual system research. The purpose of this study was to investigate the localization of calcium-binding proteins (CBPs) in the visual cortex of the Mongolian gerbil (Meriones unguiculatus). We also compared the labeling of CBPs to those of gamma-aminobutyric acid (GABA)- and nitric oxide synthase (NOS)-containing neurons.
Material and methods:
The study was conducted on twelve adult Mongolian gerbils (3-4 months old). We used horseradish peroxidase immunocytochemistry and two-color fluorescence immunocytochemistry with conventional and confocal microscopy to assess CBPs localization in the visual cortex.
Results:
The highest density of calbindin-D28K (CB)- (34.18%) and parvalbumin (PV)-IR (37.51%) neurons was found in layer V, while the highest density of calretinin (CR)-IR (33.85%) neurons was found in layer II. The CB- (46.99%), CR- (44.88%), and PV-IR (50.17%) neurons mainly displayed a multipolar round/oval morphology. Two-color immunofluorescence revealed that only 16.67%, 14.16%, and 39.91% of the CB-, CR-, and PV-IR neurons, respectively, contained GABA. In addition, none of the CB-, CR-, and PV-IR neurons contained NOS.
Conclusions:
Our findings indicate that CB-, CR-, and PV-containing neurons in the Mongolian gerbil visual cortex are distributed abundantly and distinctively in specific layers and in a small population of GABAergic neurons but are limited to subpopulations that do not express NOS. These data provide a basis for the potential roles of CBP-containing neurons in the gerbil visual cortex.
... Serotonergic varicosities are likely to be fluid structures, as suggested by experimental studies of mice and Drosophila with elevated serotonin levels (Daubert et al., 2010), a rat model of epilepsy (Maia et al., 2019), and analyses of varicosities in other neurotransmitter systems. Specifically, the appearance of varicosities may reflect the current state of the fiber segment, the effects of its local microenvironment, and purely stochastic fluctuations (Hellwig et al., 1994;Hatada et al., 1999;Shepherd et al., 2002;Gu, 2021;Ma et al., 2022). Notably, the dynamics of varicosities on the same fiber are likely to be correlated, necessitating unambiguous discrimination among individual fibers that often cross and intertwine in the same spatial location. ...
The self-organization of the serotonergic matrix, a massive axon meshwork in all vertebrate brains, is driven by the structural and dynamical properties of its constitutive elements. Each of these elements, a single serotonergic axon (fiber), has a unique trajectory and can be supported by a soma that executes one of many available transcriptional programs. It necessitates the development of specialized methods for single-fiber analyses, both at the experimental and theoretical levels. We developed an integrated system that facilitates experimental isolation of single serotonergic fibers in brain tissue, including regions with high fiber densities, and demonstrated the potential of their quantitative analyses based on stochastic modeling. Single fibers were visualized using two transgenic mouse models, one of which is the first implementation of the Brainbow toolbox in this system. The trajectories of serotonergic fibers were automatically traced in the three spatial dimensions with a novel algorithm, and their properties were captured with a single parameter associated with the directional von Mises-Fisher probability distribution. The system represents an end-to-end workflow that can be imported into various studies, including those investigating serotonergic dysfunction in brain disorders. It also supports new research directions inspired by single-fiber analyses in the serotonergic matrix, including supercomputing simulations and modeling in physics.
... During trauma, rapid deformation causes microtubules, the internal support structures of axons involved in axonal transport, to become brittle and periodically snap along their length [64]. This results in the accumulation of essential organelles and proteins (for example, amyloid precursor protein) that would normally be trafficked back to the cell body [38,65], leading to the formation of axonal varicosities (swellings) along the length of an axon in a 'string of beads' formation [66]. These swellings can lead to the formation of retraction bulbs and disconnection of the axon, although some axons can spontaneously recover [66][67][68]. ...
... This results in the accumulation of essential organelles and proteins (for example, amyloid precursor protein) that would normally be trafficked back to the cell body [38,65], leading to the formation of axonal varicosities (swellings) along the length of an axon in a 'string of beads' formation [66]. These swellings can lead to the formation of retraction bulbs and disconnection of the axon, although some axons can spontaneously recover [66][67][68]. Equally, axons that initially appear undamaged post-TBI, with no interruption of axonal transport mechanisms, may degenerate later as a result of secondary cascade mechanisms [69], with microtubule networks and myelin fragments potentially playing a role in the survival or destruction of the axon [70,71]. Axonal varicosities either resolve or become axonal bulbs within a short period of time after trauma (around two hours [72]), so their presence at time points distal from the initial injury is indicative of an on-going degenerative process, possibly as a result of continued neuroinflammation [73]. ...
An estimated sixty-nine million people sustain a traumatic brain injury each year. Trauma to the brain causes the primary insult and initiates a secondary biochemical cascade as part of the immune and reparative response to injury. The secondary cascade, although a normal physiological response, may also contribute to ongoing neuroinflammation, oxidative stress and axonal injury, continuing in some cases years after the initial insult. In this review, we explain some of the biochemical mechanisms of the secondary cascade and their potential deleterious effects on healthy neurons including secondary cell death. The second part of the review focuses on the role of micronutrients to neural mechanisms and their potential reparative effects with regards to the secondary cascade after brain injury. The biochemical response to injury, hypermetabolism and excessive renal clearance of nutrients after injury increases the demand for most vitamins. Currently, most research in the area has shown positive outcomes of vitamin supplementation after brain injury, although predominantly in animal (murine) models. There is a pressing need for more research in this area with human participants because vitamin supplementation post-trauma is a potential cost-effective adjunct to other clinical and therapeutic treatments. Importantly, traumatic brain injury should be considered a lifelong process and better evaluated across the lifespan of individuals who experience brain injury.
... In this study, we observed axonal varicosities and reduced axonal elongation in CTX and SPG5 iPSC-derived neurons. Increased axonal varicosities and swellings are characteristic pathologies caused by impaired axonal transport and accumulated organelles, which has also been observed in models of common forms of HSP [36,43,56]. These findings suggest that impaired vesicular transport along axons as well as perturbed axonal extension can occur downstream of cholesterol and bile acid metabolic pathway defects [13,57] as a common pathomechanism for axonal degeneration in both CTX and SPG5. ...
Background
Biallelic mutations in CYP27A1 and CYP7B1 , two critical genes regulating cholesterol and bile acid metabolism, cause cerebrotendinous xanthomatosis (CTX) and hereditary spastic paraplegia type 5 (SPG5), respectively. These rare diseases are characterized by progressive degeneration of corticospinal motor neuron axons, yet the underlying pathogenic mechanisms and strategies to mitigate axonal degeneration remain elusive.
Methods
To generate induced pluripotent stem cell (iPSC)-based models for CTX and SPG5, we reprogrammed patient skin fibroblasts into iPSCs by transducing fibroblast cells with episomal vectors containing pluripotency factors. These patient-specific iPSCs, as well as control iPSCs, were differentiated into cortical projection neurons (PNs) and examined for biochemical alterations and disease-related phenotypes.
Results
CTX and SPG5 patient iPSC-derived cortical PNs recapitulated several disease-specific biochemical changes and axonal defects of both diseases. Notably, the bile acid chenodeoxycholic acid (CDCA) effectively mitigated the biochemical alterations and rescued axonal degeneration in patient iPSC-derived neurons. To further examine underlying disease mechanisms, we developed CYP7B1 knockout human embryonic stem cell (hESC) lines using CRISPR-cas9-mediated gene editing and, following differentiation, examined hESC-derived cortical PNs. Knockout of CYP7B1 resulted in similar axonal vesiculation and degeneration in human cortical PN axons, confirming a cause-effect relationship between gene deficiency and axonal degeneration. Interestingly, CYP7B1 deficiency led to impaired neurofilament expression and organization as well as axonal degeneration, which could be rescued with CDCA, establishing a new disease mechanism and therapeutic target to mitigate axonal degeneration.
Conclusions
Our data demonstrate disease-specific lipid disturbances and axonopathy mechanisms in human pluripotent stem cell-based neuronal models of CTX and SPG5 and identify CDCA, an established treatment of CTX, as a potential pharmacotherapy for SPG5. We propose this novel treatment strategy to rescue axonal degeneration in SPG5, a currently incurable condition.
... Between 12 and 24 h postinjury, indeed, delayed (or secondary) axotomy occurs [32]. The distal, isolated portion of the axon undergoes Wallerian degeneration, while the proximal segment retains the ability to regenerate [33]. Multiple factors are involved in this process, but the key role is played by the neurofilament protein subunits, with the misalignment of the filaments, the disruption of axoplasmic flow, and swelling [34]. ...
Wound healing is characterized by the formation of a granulation tissue consisting of inflammatory cells, newly formed blood vessels, and fibroblasts embedded in a loose collagenous extracellular matrix. Tumors behave as wounds that fail to heal. Neuronal loss in neurodegenerative disease is associated with the synthesis and release of new components of the extracellular matrix by activated fibroblasts and astrocytes. This condition is responsible for a perpetuation of the wound healing state and constitutes a condition very similar to that which occurs during tumor progression. The aim of this article is to emphasize and compare the role of wound healing in two different pathological conditions, namely tumor growth and central nervous system neurodegenerative diseases. Both are conditions in which wounds fail to heal, as occurs in physiological conditions.
... They have been previously reported to be potential axonal branching points 55 and are presently viewed to be highly relevant in neural injury and neurodegeneration. 56 Figure 4C). We further calculated the surface DAT fraction using the Henderson-Hasselbalch equation and found that axonal shafts and boutons exhibited a similar $75% surface fraction of DAT and similar acidity for DAT containing vesicles ( Figures 4D and 4E). ...
Cocaine acts by inhibiting plasma membrane dopamine transporter (DAT) function as well as altering its surface expression. The precise manner and mechanism by which cocaine regulates DAT trafficking, especially at neuronal processes, are poorly understood. In this study, we engineered and validated the use of DAT-pHluorin for studying DAT localization and its dynamic trafficking at neuronal processes of cultured mouse midbrain neurons. We show that unlike neuronal soma and dendrites, which contain majority of the DATs in weakly acidic intracellular compartments, axonal DATs at both shafts and boutons are primarily (75%) localized to the plasma membrane, while large varicosities contain abundant intracellular DAT within acidic intracellular structures. We also demonstrate that cocaine exposure leads to a Synaptojanin1-sensitive DAT internalization process followed by membrane reinsertion that lasts for days. Thus, our study reveals the previously unknown dynamics and molecular regulation for cocaine-regulated DAT trafficking in neuronal processes.
... Early studies in other systems have suggested that varicosities can form directly from "stopped" growth cones (Hatada et al., 1999), and that their distribution can be described by random point-processes such as the Poison process (Hellwig et al., 1994). More recent studies have shown that varicosities strongly respond to their mechanical and biological environment (Shepherd et al., 2002;Ma et al., 2022) and are generally plastic, including pathological states (Gu, 2021). In the mouse brain, serotonergic axons appear to undergo a developmental transition from "dot-like" enlargements, with virtually undetectable connections, to a smooth morphology by the end of the first developmental month (Maddaloni et al., 2017). ...
Vertebrate brains have a dual structure, composed of (i) axons that can be well-captured with graph-theoretical methods and (ii) axons that form a dense matrix in which neurons with precise connections operate. A core part of this matrix is formed by axons (fibers) that store and release 5-hydroxytryptamine (5-HT, serotonin), an ancient neurotransmitter that supports neuroplasticity and has profound implications for mental health. The self-organization of the serotonergic matrix is not well understood, despite recent advances in experimental and theoretical approaches. In particular, individual serotonergic axons produce highly stochastic trajectories, fundamental to the construction of regional fiber densities, but further advances in predictive computer simulations require more accurate experimental information. This study examined single serotonergic axons in culture systems (co-cultures and monolayers), by using a set of complementary high-resolution methods: confocal microscopy, holotomography (refractive index-based live imaging), and super-resolution (STED) microscopy. It shows that serotonergic axon walks in neural tissue may strongly reflect the stochastic geometry of this tissue and it also provides new insights into the morphology and branching properties of serotonergic axons. The proposed experimental platform can support next-generation analyses of the serotonergic matrix, including seamless integration with supercomputing approaches.
... ± 0.17) and dendrites (pH=6.94 ± 0.22) (Fig 3F). 10 11 Varicosities are distinct axonal sites that contain DAT in acidic intracellular organelles 12 13 The relative distribution of intracellular versus plasma membrane DAT at rest in different presently viewed to be highly relevant in neural injury and neurodegeneration (Gu 2021). We found that only varicosities but not axonal shafts or boutons exhibited a robust NH4Cl response 1 ( Fig. 4B-C). ...
The dopamine transporter (DAT) mediated DA reuptake is a major molecular mechanism for termination of dopaminergic signaling in the brain. Psychoactive substances such as cocaine act by inhibition of plasma membrane DAT function as well as by altering its expression. The precise manner and mechanism by which cocaine regulates DAT trafficking, especially at neuronal processes, are poorly understood. We have now engineered a novel pH-sensitive reporter for DAT by conjugating pHluorin to the second exofacial loop of human DAT. We show that DAT-pHluorin can be used to study DAT localization and its dynamic trafficking at neuronal processes. Using DAT-pHluorin we show that unlike neuronal soma and dendrites, which contain majority of the DATs in weakly acidic intracellular compartments, axonal DATs at both shafts and boutons are primarily (75%) localized to the plasma membrane, while varicosities contain abundant intracellular DAT within acidic intracellular structures. Using this novel reporter, we show, for the first time, that cocaine exposure leads to a brief DAT internalization followed by membrane reinsertion that lasts for days. We further show that the cocaine-induced DAT trafficking is sensitive to the activities of Synaptojanin1 phosphatase. Thus, our study using the newly engineered DAT optical reporter reveals the previously unknown dynamics and molecular regulation for cocaine-regulated DAT trafficking in neuronal processes.
