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| The majority of Purkinje cell torpedoes contain neurofilament (NF). (A) Sample image showing Purkinje cells and their axons (green) and neurofilament (red). Co-labeling appears yellow. Most torpedoes are neurofilament (NF) positive, although some are not. Inset shows even neighboring torpedoes on the same axon can be NF+ and NF−. Scale bar in left image, 20 µm; Scale bar in inset, 5 µm. (B) Quantification of NF+ (white bars) and NF− (orange bars) at P11 and P30. A sizeable minority of torpedoes are NF−, especially at P11, suggesting that there may be two populations of developmental torpedoes. Significance determined by Mann-Whitney U test, ns P > 0.05.
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Information is carried out of the cerebellar cortical microcircuit via action potentials propagated along Purkinje cell axons. In several human neurodegenerative diseases, focal axonal swellings on Purkinje cells – known as torpedoes – have been associated with Purkinje cell loss. Interestingly, torpedoes are also reported to appear transiently dur...
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... wondered whether developmental torpedoes also contained neurofilament. To address this, we labeled neurofilament 200 kD (NF) and counted the torpedoes that were positive (NF+) and negative (NF−) at both P11 and P30 (Figure 6A). At the peak of Purkinje cell torpedo density at P11, although the majority of Purkinje cell axon torpedoes were NF+ (51/80, or 64% of torpedoes), there was a significant fraction that were not (29/80, or 36%; Figure 6B). ...
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... address this, we labeled neurofilament 200 kD (NF) and counted the torpedoes that were positive (NF+) and negative (NF−) at both P11 and P30 (Figure 6A). At the peak of Purkinje cell torpedo density at P11, although the majority of Purkinje cell axon torpedoes were NF+ (51/80, or 64% of torpedoes), there was a significant fraction that were not (29/80, or 36%; Figure 6B). Sometimes neurofilament positive and negative torpedoes were next to each other on the same axon ( Figure 6A, inset), suggesting that these two types of torpedoes may have distinct functions that are locally determined. ...
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... the peak of Purkinje cell torpedo density at P11, although the majority of Purkinje cell axon torpedoes were NF+ (51/80, or 64% of torpedoes), there was a significant fraction that were not (29/80, or 36%; Figure 6B). Sometimes neurofilament positive and negative torpedoes were next to each other on the same axon ( Figure 6A, inset), suggesting that these two types of torpedoes may have distinct functions that are locally determined. Consistent with this, NF+ torpedoes were significantly larger than NF− torpedoes (NF+ length: 7.6 ± 0.3 µm; NF− length: 6.1 ± 0.2 µm; significantly different, P = 0.0001; NF+ width: 3.4 ± 0.2 µm; NF− width 2.7 ± 0.1 µm; significantly different, P = 0.0007). ...
Citations
... In addition, the morphogenesis of PCs starts to present some defects, with the upregulation of the neurofilament light chain gene (Nefl). Neurofilaments are cytoskeletal components enriched at PC axonal "torpedoes" or focal swellings, and, while their function is poorly understood, they seem to be a developmental characteristic which peaks at P11 (Ljungberg et al., 2016). This could signify that, during IH, PCs modify their phenotype, and later, their connection profile. ...
Apnea of prematurity (AOP) affects more than 50% of preterm infants and leads to perinatal intermittent hypoxia (IH) which is a major cause of morbimortality worldwide. At birth, the human cerebellar cortex is still immature, making it vulnerable to perinatal events. Additionally, studies have shown a correlation between cerebellar functions and the deficits observed in children who have experienced AOP. Yet, the cerebellar alterations underpinning this link remain poorly understood. To gain insight into the involvement of the cerebellum in perinatal hypoxia-related consequences, we developed a mouse model of AOP. Our previous research has revealed that IH induces oxidative stress in the developing cerebellum, as evidenced by the over-expression of genes involved in reactive oxygen species production and the under-expression of genes encoding antioxidant enzymes. These changes suggest a failure of the defense system against oxidative stress and could be responsible for neuronal death in the cerebellum.
Building upon these findings, we conducted a transcriptomic study of the genes involved in the processes that occur during cerebellar development. Using real-time PCR, we analyzed the expression of these genes at different developmental stages and in various cell types. This enabled us to pinpoint a timeframe of vulnerability at P8, which represents the age with the highest number of downregulated genes in the cerebellum. Furthermore, we discovered that our IH protocol affects several molecular pathways, including proliferation, migration, and differentiation. This indicates that IH can impact the development of different cell types, potentially contributing to the histological and behavioral deficits observed in this model. Overall, our data strongly suggest that the cerebellum is highly sensitive to IH, and provide valuable insights into the cellular and molecular mechanisms underlying AOP. In the long term, these findings may contribute to the identification of novel therapeutic targets for improving the clinical management of this prevalent pathology.
... The samples were prepared from eight slices across the brain regions of each mouse. The 25 Â 25 mm region of interest was outlined in the cerebellum around torpedoes and calculated as previously described (Ljungberg et al., 2016). PV 1 and choline O-acetyltransferase-positive (ChAT 1 ) neurons were counted in eight sections of the lumbar spinal cord (L1-L5) from Tfr1-cKO mice and wild-type (WT) littermates. ...
Hereditary spastic paraplegia (HSP) is a severe neurodegenerative movement disorder, the underlying pathophysiology of which remains poorly understood. Mounting evidence has suggested that iron homeostasis dysregulation can lead to motor function impairment. However, whether deficits in iron homeostasis are involved in the pathophysiology of HSP remains unknown. To address this knowledge gap, we focused on parvalbumin-positive (PV ⁺ ) interneurons, a large category of inhibitory neurons in the central nervous system, which play a critical role in motor regulation. The PV ⁺ interneuron-specific deletion of the gene encoding transferrin receptor 1 (TFR1)—a key component of the neuronal iron uptake machinery—induced severe progressive motor deficits in both male and female mice. In addition, we observed skeletal muscle atrophy, axon degeneration in the spinal cord dorsal column, and alterations in the expression of HSP-related proteins in male mice with Tfr1 deletion in the PV ⁺ interneurons. These phenotypes were highly consistent with the core clinical features of HSP cases. Furthermore, the effects on motor function induced by Tfr1 ablation in PV ⁺ interneurons were mostly concentrated in the dorsal spinal cord; however, iron repletion partly rescued the motor defects and axon loss seen in both sexes of conditional Tfr1 mutant mice. Our study describes a new mouse model for mechanistic and therapeutic studies relating to HSP and provides novel insights into iron metabolism in spinal cord PV ⁺ interneurons and its role in the regulation of motor functions.
Significance
Iron is crucial for neuronal functioning. Mounting evidence suggests that iron homeostasis dysregulation can induce motor function deficits. Transferrin receptor 1 (TFR1) is thought to be the key component in neuronal iron uptake. We found that deletion of Tfr1 in parvalbumin-positive (PV ⁺ ) interneurons in mice induced severe progressive motor deficits, skeletal muscle atrophy, axon degeneration in the spinal cord dorsal column, and alterations in the expression of hereditary spastic paraplegia (HSP)-related proteins. These phenotypes were highly consistent with the core clinical features of HSP cases and partly rescued by iron repletion. This study describes a new mouse model for the study of HSP and provides novel insights into iron metabolism in spinal cord PV ⁺ interneurons.
... As for SCA6, mechanistic evidence suggests a link between the disease hallmark of Purkinje cell torpedoes and myelination. Torpedoes are focal swellings or spheroids of an axon and are thought to increase in number on Purkinje cells as a response to cerebellar injury [79][80][81]. Increased torpedo formation has been demonstrated in essential tremor [82], a cerebellum-linked condition, as well as SCA6 [83]. One study evaluated the number of torpedoes on Purkinje cells in transgenic SCA6 84Q/84Q mice [80]. ...
... Increased torpedo formation has been demonstrated in essential tremor [82], a cerebellum-linked condition, as well as SCA6 [83]. One study evaluated the number of torpedoes on Purkinje cells in transgenic SCA6 84Q/84Q mice [80]. This study found the highest number of torpedoes at P11, most of which exist on myelinated axons. ...
Human studies, in combination with animal and cellular models, support glial cells as both major contributors to neurodegenerative diseases and promising therapeutic targets. Among glial cells, oligodendrocytes and Schwann cells are the myelinating glial cells of the central and peripheral nervous system, respectively. In this review, we discuss the contributions of these central and peripheral myelinating glia to the pathomechanisms of polyglutamine (polyQ) spinocerebellar ataxia (SCA) types 1, 2, 3, 6, 7, and 17. First, we highlight the function of oligodendrocytes in healthy conditions and how they are disrupted in polyQ SCA patients and diseased model systems. We then cover the role of Schwann cells in peripheral nerve function and repair as well as their possible role in peripheral neuropathy in polyQ SCAs. Finally, we discuss potential polyQ SCA therapeutic interventions in myelinating glial.
... Focal axonal swellings arise in several regions of the central nervous system (CNS) in a number of neurodegenerative disorders and in traumatic brain injury (Trapp et al., 1998;Nikić et al., 2011;Sorbara et al., 2014;Jin et al., 2015;Maia et al., 2015). In these cases, the axonal swellings on cerebellar Purkinje cells-known as torpedoes-are associated with Purkinje cell loss (Louis et al., 2009(Louis et al., , 2014Maia et al., 2015;Ljungberg., 2016;Gionco et al., 2021). Although there are many causes of Purkinje axonal swellings, there are common morphological features in Purkinje axons in the mouse models with perturbations of PNJs, described in the following. ...
... The focal axonal swellings in Purkinje cells are found in various pathological conditions as well as during development. A study by Lang-Ouellette and others recently reported that Purkinje axonal swellings in healthy young mice enhance action potential fidelity and cerebellar function (Ljungberg et al., 2016;Lans-Quellette et al., 2021). However, the density of intracellular organelles in the axonal swellings did not change compared to control axons (Lans-Quellette et al., 2021), suggesting that the Purkinje axonal swellings in healthy development and disease models differ in their subcellular composition. ...
In vertebrates, a high density of voltage-gated Na+ channel at nodes of Ranvier and of voltage-gated K+ channel at juxtaparanodes is necessary for rapid propagation of action potential, that is, for saltatory conduction in myelinated axons. Myelin loops attach to the axonal membrane and form paranodal axoglial junctions (PNJs) at paranodes adjacent to nodes of Ranvier. There is growing evidence that the PNJs contribute to axonal homeostasis in addition to their roles as lateral fences that restrict the location of nodal axolemmal proteins for effective saltatory conduction. Perturbations of PNJs, as in specific PNJ protein knockouts as well as in myelin lipid deficient mice, result in internodal axonal alterations, even if their internodal myelin is preserved. Here we review studies showing that PNJs play crucial roles in the myelinated axonal homeostasis. The present evidence points to two functions in particular: 1) PNJs facilitate axonal transport of membranous organelles as well as cytoskeletal proteins; and 2) they regulate the axonal distribution of type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) in cerebellar Purkinje axons. Myelinated axonal homeostasis depends among others on the state of PNJs, and consequently, a better understanding of this dependency may contribute to the clarification of CNS disease mechanisms and the development of novel therapies.
... Torpedoes in the Purkinje cell axons, like we observed in the NEB-cases, are reported in a wide range of diseases, such as spinocerebellar ataxias in humans [33] and abiotrophies in several dog breeds [34]. Additionally, torpedoes has been reported from transgenic ataxic mice expressing constitutive active Rac1 in Purkinje cells [35]. ...
A number of inherited ataxias is known in humans, with more than 250 loci implicated, most of which are included in human ataxia screening panels. Anecdotally, cases of ataxia in the Norwegian elkhound black have been known for the last 40 years. Affected puppies from three litters were clinically and neurologically examined, and postmortem samples were collected for morphological studies, including ultrastructural analyses. The puppies displayed vestibulocerebellar neurological signs and had degenerative histopathological alterations in cerebellum and brain stem. Three affected dogs, each from different litters, as well as both parents and one healthy littermate from each litter, were whole genome sequenced. Through variant calling we discovered a disease-associated 1 bp deletion in HACE1 (CFA12), resulting in a frameshift at codon 333 and a premature stop codon at codon 366. The perfect association combined with the predicted significant molecular effect, strongly suggest that we have found the causative mutation for Norwegian elkhound black ataxia. We have identified a novel candidate gene for ataxia where dogs can serve as a spontaneous model for improved understanding of ataxia, also in human.
... Since Purkinje cell axons carry information out of the cerebellar cortex, changes in the structure of their axons could impact cerebellar function dramatically. Purkinje cell axonal swellings appear transiently during cerebellar development 3,4 and are observed during normal aging 5 , including in healthy human samples 6,7 . These data suggest that axonal swellings play a physiological role in the brain. ...
... Action potential propagation is enhanced in axons with swellings. Purkinje cell axonal swellings are present in healthy developing rodents 4 and are also observed in several neurodegenerative diseases [8][9][10][11] . However, functional measurements of the impact of axonal swellings on axons have been lacking. ...
... Since computational models suggest that axonal swellings increase axonal failure rates 13 , we monitored action potential failures in Purkinje cells with and without focal axonal swellings (Fig. 1b). Approximately 30% of axons display swellings at this age 4 , and the vast majority of these were single swellings within the granule cell layer (98.7%; Supplementary Fig. 1), which we targeted for our recordings of axonal swellings. ...
Axonal plasticity allows neurons to control their output, which critically determines the flow of information in the brain. Axon diameter can be regulated by activity, yet how morphological changes in an axon impact its function remains poorly understood. Axonal swellings have been found on Purkinje cell axons in the cerebellum both in healthy development and in neurodegenerative diseases, and computational models predicts that axonal swellings impair axonal function. Here we report that in young Purkinje cells, axons with swellings propagated action potentials with higher fidelity than those without, and that axonal swellings form when axonal failures are high. Furthermore, we observed that healthy young adult mice with more axonal swellings learn better on cerebellar-related tasks than mice with fewer swellings. Our findings suggest that axonal swellings underlie a form of axonal plasticity that optimizes the fidelity of action potential propagation in axons, resulting in enhanced learning. Axonal swellings have been found on Purkinje cell axons in the cerebellum both during development and disease. The authors show that axons with swellings propagate action potentials with higher fidelity than those without and that mice with more axonal swellings learn cerebellar-related tasks better.
... Several other factors likely contribute to the increased latency of action potentials arising from axonal stimulation. Purkinje cell axons are myelinated (Ljungberg et al., 2016) and action potentials travel between nodes of Ranvier in the axon. However, given that internodal spacing ranges between 60 and 260 µm (Clark et al., 2005), the area of focal photostimulation is likely to only occasionally overlap with a node of Ranvier. ...
Optogenetics is a state-of-the-art tool for interrogating neural circuits. In the cerebellum, Purkinje cells serve as the sole output of the cerebellar cortex where they synapse on neurons in the deep cerebellar nuclei (DCN). To investigate the properties of this synaptic connection, we sought to elicit time-locked single action potentials from Purkinje cell axons. Using optical stimulation of channelrhodopsin-2 (ChR2)-expressing Purkinje cells combined with patch-clamp recordings of Purkinje cells and DCN neurons in acute cerebellar slices, we determine the photostimulation parameters required to elicit single time-locked action potentials from Purkinje cell axons. We show that axons require longer light pulses than somata do to elicit single action potentials and that Purkinje cell axons are also more susceptible to light perturbations. We then demonstrate that these empirically determined photostimulation parameters elicit time-locked synaptic currents from postsynaptic cells in the DCN. Our results highlight the importance of optimizing optogenetic stimulation conditions to interrogate synaptic connections.
... Importantly, degeneration of neuronal synapses and processes became progressively more severe with age, culminating in neuronal loss. Inactivation of PI31 in MNs and PCs recapitulated the progressive neuropathology and motor dysfunction of previously described mouse models of ALS and ataxia, respectively, and was reminiscent of the severe behavioral and anatomical defects associated with human spinal MN and PC neurodegenerative diseases (54)(55)(56)(57)(58). Collectively, this work establishes a critical role for PI31 and protein degradation in the maintenance of neuronal architecture, circuitry, and function. ...
Significance
The conserved proteasome-binding protein PI31 serves as an adapter to couple proteasomes with cellular motors to mediate their transport to distal tips of neurons where protein breakdown occurs. We generated global and conditional PI31 knockout mouse strains and show that this protein is required for protein homeostasis, and that its conditional inactivation in neurons disrupts synaptic structures and long-term survival. This work establishes a critical role for PI31 and local protein degradation in the maintenance of neuronal architecture, circuitry, and function. Because mutations in the PI31 pathway cause neurodegenerative diseases in humans, reduced PI31 activity may contribute to the etiology of these diseases.
... Cacna1a/Q84 knockin mice were generated by introducing the mouse gene with 84 polyglutamine repeats at the mouse locus, causing cytoplasmic inclusions in Purkinje cells and rotorod deficits [70]. Cacna1a/Q84 knockins also displayed Purkinje cell axon anomalies in the form of torpedoes as found in SCA6 patients [144]. Cacna1a/Q84 knockin mice exhibited late-onset loss of Purkinje cell number but early-onset increase in foot-slips on the stationary beam and decreased latencies before falling off the rotorod despite the absence of any change in stride length or stance width [145]. ...
Chance discovery of spontaneous mutants with atrophy of the cerebellar cortex has unearthed genes involved in optimizing motor coordination. Rotorod, stationary beam, and suspended wire tests are useful in delineating behavioral phenotypes of spontaneous mutants with cerebellar atrophy such as Grid2Lc, Grid2ho, Rorasg, Agtpbp1pcd, Relnrl, and Dab1scm. Likewise, transgenic or null mutants serving as experimental models of spinocerebellar ataxia (SCA) are phenotyped with the same tests. Among experimental models of autosomal dominant SCA, rotorod deficits were reported in SCA1 to 3, SCA5 to 8, SCA14, SCA17, and SCA27 and stationary beam deficits in SCA1 to 3, SCA5, SCA6, SCA13, SCA17, and SCA27. Beam tests are sensitive to experimental therapies of various kinds including molecules affecting glutamate signaling, mesenchymal stem cells, anti-oligomer antibodies, lentiviral vectors carrying genes, interfering RNAs, or neurotrophic factors, and interbreeding with other mutants.
... Our data suggest that basket cell terminals could serve a role in maintaining axonal integrity as we frequently observed swellings of Purkinje cell axons proximal to the AIS in Neurod2 −/− mutants. Axonal swellings ('torpedoes') are a common feature of various neurodegenerative diseases with cerebellar involvement, but also occur in the developing mouse cerebellum and peak at P11 56,57 . Axonal swellings in Neurod2 −/− mutants are not merely a consequence of abolished inhibition, as the removal of GABA A receptor-mediated synaptic inhibition in Purkinje cells causes no corresponding axonal pathology 50 . ...
The cerebellar cortex is involved in the control of diverse motor and non-motor functions. Its principal circuit elements are the Purkinje cells that integrate incoming excitatory and local inhibitory inputs and provide the sole output of the cerebellar cortex. However, the transcriptional control of circuit assembly in the cerebellar cortex is not well understood. Here, we show that NeuroD2, a neuronal basic helix-loop-helix (bHLH) transcription factor, promotes the postnatal survival of both granule cells and molecular layer interneurons (basket and stellate cells). However, while NeuroD2 is not essential for the integration of surviving granule cells into the excitatory circuit, it is required for the terminal differentiation of basket cells. Axons of surviving NeuroD2-deficient basket cells follow irregular trajectories and their inhibitory terminals are virtually absent from Purkinje cells in Neurod2 mutants. As a result inhibitory, but not excitatory, input to Purkinje cells is strongly reduced in the absence of NeuroD2. Together, we conclude that NeuroD2 is necessary to instruct a terminal differentiation program in basket cells that regulates targeted axon growth and inhibitory synapse formation. An imbalance of excitation and inhibition in the cerebellar cortex affecting Purkinje cell output may underlay impaired adaptive motor learning observed in Neurod2 mutants.
