Dual spatio-temporal regulation of axon growth and microtubule dynamics by RhoA signaling pathways.

RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localizes in growth cones of undifferentiated neurites, while in developing axons it displays a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevents axon initiation but has no effect on elongation, while formin inhibition reduces axon extension without significantly altering initial outgrowth. Besides, RhoA-mDia promotes axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK that restrains growth cone microtubule assembly and protrusion.

Integrins protect sensory neurons in models of paclitaxel-induced peripheral sensory neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect from cancer treatment with no known method for prevention or cure in clinics. CIPN often affects unmyelinated nociceptive sensory terminals. Despite the high prevalence, molecular and cellular mechanisms that lead to CIPN are still poorly understood. Here, we used a genetically tractable Drosophila model and primary sensory neurons isolated from adult mouse to examine the mechanisms underlying CIPN and identify protective pathways. We found that chronic treatment of Drosophila larvae with paclitaxel caused degeneration and altered the branching pattern of nociceptive neurons, and reduced thermal nociceptive responses. We further found that nociceptive neuron-specific overexpression of integrins, which are known to support neuronal maintenance in several systems, conferred protection from paclitaxel-induced cellular and behavioral phenotypes. Live imaging and superresolution approaches provide evidence that paclitaxel treatment causes cellular changes that are consistent with alterations in endosome-mediated trafficking of integrins. Paclitaxel-induced changes in recycling endosomes precede morphological degeneration of nociceptive neuron arbors, which could be prevented by integrin overexpression. We used primary dorsal root ganglia (DRG) neuron cultures to test conservation of integrin-mediated protection. We show that transduction of a human integrin ß-subunit 1 also prevented degeneration following paclitaxel treatment. Furthermore, endogenous levels of surface integrins were decreased in paclitaxel-treated mouse DRG neurons, suggesting that paclitaxel disrupts recycling in vertebrate sensory neurons. Altogether, our study supports conserved mechanisms of paclitaxel-induced perturbation of integrin trafficking and a therapeutic potential of restoring neuronal interactions with the extracellular environment to antagonize paclitaxel-induced toxicity in sensory neurons.

Pathogenic role of delta 2 tubulin in bortezomib-induced peripheral neuropathy.

The pathogenesis of chemotherapy-induced peripheral neuropathy (CIPN) is poorly understood. Here, we report that the CIPN-causing drug bortezomib (Bort) promotes delta 2 tubulin (D2) accumulation while affecting microtubule stability and dynamics in sensory neurons in vitro and in vivo and that the accumulation of D2 is predominant in unmyelinated fibers and a hallmark of bortezomib-induced peripheral neuropathy (BIPN) in humans. Furthermore, while D2 overexpression was sufficient to cause axonopathy and inhibit mitochondria motility, reduction of D2 levels alleviated both axonal degeneration and the loss of mitochondria motility induced by Bort. Together, our data demonstrate that Bort, a compound structurally unrelated to tubulin poisons, affects the tubulin cytoskeleton in sensory neurons in vitro, in vivo, and in human tissue, indicating that the pathogenic mechanisms of seemingly unrelated CIPN drugs may converge on tubulin damage. The results reveal a previously unrecognized pathogenic role for D2 in BIPN that may occur through altered regulation of mitochondria motility.

RNA methyltransferase NSun2 deficiency promotes neurodegeneration through epitranscriptomic regulation of tau phosphorylation.

Epitranscriptomic regulation adds a layer of post-transcriptional control to brain function during development and adulthood. The identification of RNA-modifying enzymes has opened the possibility of investigating the role epitranscriptomic changes play in the disease process. NOP2/Sun RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases able to methylate mammalian non-coding RNAs. NSun2 loss of function due to autosomal-recessive mutations has been associated with neurological abnormalities in humans. Here, we show NSun2 is expressed in adult human neurons in the hippocampal formation and prefrontal cortex. Strikingly, we unravel decreased NSun2 protein expression and an increased ratio of pTau/NSun2 in the brains of patients with Alzheimer's disease (AD) as demonstrated by Western blotting and immunostaining, respectively. In a well-established Drosophila melanogaster model of tau-induced toxicity, reduction of NSun2 exacerbated tau toxicity, while overexpression of NSun2 partially abrogated the toxic effects. Conditional ablation of NSun2 in the mouse brain promoted a decrease in the miR-125b m6A levels and tau hyperphosphorylation. Utilizing human induced pluripotent stem cell (iPSC)-derived neuronal cultures, we confirmed NSun2 deficiency results in tau hyperphosphorylation. We also found that neuronal NSun2 levels decrease in response to amyloid-beta oligomers (AßO). Notably, AßO-induced tau phosphorylation and cell toxicity in human neurons could be rescued by overexpression of NSun2. Altogether, these results indicate that neuronal NSun2 deficiency promotes dysregulation of miR-125b and tau phosphorylation in AD and highlights a novel avenue for therapeutic targeting.

Tubulin tyrosination regulates synaptic function and is disrupted in Alzheimer's disease.

Microtubules play fundamental roles in the maintenance of neuronal processes and in synaptic function and plasticity. While dynamic microtubules are mainly composed of tyrosinated tubulin, long-lived microtubules contain detyrosinated tubulin, suggesting that the tubulin tyrosination/detyrosination cycle is a key player in the maintenance of microtubule dynamics and neuronal homeostasis, conditions that go awry in neurodegenerative diseases. In the tyrosination/detyrosination cycle, the C-terminal tyrosine of α-tubulin is removed by tubulin carboxypeptidases and re-added by tubulin tyrosine ligase (TTL). Here we show that TTL heterozygous mice exhibit decreased tyrosinated microtubules, reduced dendritic spine density and both synaptic plasticity and memory deficits. We further report decreased TTL expression in sporadic and familial Alzheimer's disease, and reduced microtubule dynamics in human neurons harbouring the familial APP-V717I mutation. Finally, we show that synapses visited by dynamic microtubules are more resistant to oligomeric amyloid-ß peptide toxicity and that expression of TTL, by restoring microtubule entry into spines, suppresses the loss of synapses induced by amyloid-ß peptide. Together, our results demonstrate that a balanced tyrosination/detyrosination tubulin cycle is necessary for the maintenance of synaptic plasticity, is protective against amyloid-ß peptide-induced synaptic damage and that this balance is lost in Alzheimer's disease, providing evidence that defective tubulin retyrosination may contribute to circuit dysfunction during neurodegeneration in Alzheimer's disease.

Arfgef1 haploinsufficiency in mice alters neuronal endosome composition and decreases membrane surface postsynaptic GABAA receptors.

ARFGEF1 encodes a guanine exchange factor involved in intracellular vesicle trafficking, and is a candidate gene for childhood genetic epilepsies. To model ARFGEF1 haploinsufficiency observed in a recent Lennox Gastaut Syndrome patient, we studied a frameshift mutation (Arfgef1fs) in mice. Arfgef1fs/+ pups exhibit signs of developmental delay, and Arfgef1fs/+ adults have a significantly decreased threshold to induced seizures but do not experience spontaneous seizures. Histologically, the Arfgef1fs/+ brain exhibits a disruption in the apical lining of the dentate gyrus and altered spine morphology of deep layer neurons. In primary hippocampal neuron culture, dendritic surface and synaptic but not total GABAA receptors (GABAAR) are reduced in Arfgef1fs/+ neurons with an accompanying decrease in the number of GABAAR-containing recycling endosomes in cell body. Arfgef1fs/+ neurons also display differences in the relative ratio of Arf6+:Rab11+:TrfR+ recycling endosomes. Although the GABAAR-containing early endosomes in Arfgef1fs/+ neurons are comparable to wildtype, Arfgef1fs/+ neurons show an increase in the number of GABAAR-containing lysosomes in dendrite and cell body. Together, the altered endosome composition and decreased neuronal surface GABAAR results suggests a mechanism whereby impaired neuronal inhibition leads to seizure susceptibility.

The Aß1₋42 peptide regulates microtubule stability independently of tau.

Interference with microtubule stability by beta-amyloid peptide (Aß) has been shown to disrupt dendritic function and axonal trafficking, both early events in Alzheimer's disease. However, it is unclear whether Aß regulation of microtubule dynamics can occur independently of its action on tau. RhoA has been implicated in neurotoxicity by Aß but the mechanism by which this activation generates cytoskeletal changes is also unclear. We found that oligomeric Aß1-42 induced the formation of stable detyrosinated microtubules in NIH3T3 cells and this function resulted from the activation of a RhoA-dependent microtubule stabilization pathway regulated by integrin signaling and the formin mDia1. Induction of microtubule stability by Aß was also initiated by dimerization of APP and required caspase activity, two previously characterized regulators of neurotoxicity downstream of Aß. Finally, we found that this function was conserved in primary neurons and abolished by Rho inactivation, reinforcing a link between induction of stable detyrosinated microtubules and neuropathogenesis by Aß. Our study reveals a novel activity of Aß on the microtubule cytoskeleton that is independent of tau and associated with pathways linked to microtubule stabilization and Aß-mediated neurotoxicity.

Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin.

Neurotransmission at synapses is mediated by the fusion and subsequent endocytosis of synaptic vesicle membranes. Actin has been suggested to be required for presynaptic endocytosis but the mechanisms that control actin polymerization and its mode of action within presynaptic nerve terminals remain poorly understood. We combine optical recordings of presynaptic membrane dynamics and ultrastructural analysis with genetic and pharmacological manipulations to demonstrate that presynaptic endocytosis is controlled by actin regulatory diaphanous-related formins mDia1/3 and Rho family GTPase signaling in mouse hippocampal neurons. We show that impaired presynaptic actin assembly in the near absence of mDia1/3 and reduced RhoA activity is partly compensated by hyperactivation of Rac1. Inhibition of Rac1 signaling further aggravates impaired presynaptic endocytosis elicited by loss of mDia1/3. Our data suggest that interdependent mDia1/3-Rho and Rac1 signaling pathways cooperatively act to facilitate synaptic vesicle endocytosis by controlling presynaptic F-actin.

MFN2 coordinates mitochondria motility with α-tubulin acetylation and this regulation is disrupted in CMT2A.

Mitofusin-2 (MFN2), a large GTPase residing in the mitochondrial outer membrane and mutated in Charcot-Marie-Tooth type 2 disease (CMT2A), is a regulator of mitochondrial fusion and tethering with the ER. The role of MFN2 in mitochondrial transport has however remained elusive. Like MFN2, acetylated microtubules play key roles in mitochondria dynamics. Nevertheless, it is unknown if the α-tubulin acetylation cycle functionally interacts with MFN2. Here, we show that mitochondrial contacts with microtubules are sites of α-tubulin acetylation, which occurs through MFN2-mediated recruitment of α-tubulin acetyltransferase 1 (ATAT1). This activity is critical for MFN2-dependent regulation of mitochondria transport, and axonal degeneration caused by CMT2A MFN2 associated R94W and T105M mutations may depend on the inability to release ATAT1 at sites of mitochondrial contacts with microtubules. Our findings reveal a function for mitochondria in α-tubulin acetylation and suggest that disruption of this activity plays a role in the onset of MFN2-dependent CMT2A.

Role of tubulin post-translational modifications in peripheral neuropathy.

Peripheral neuropathy is a common disorder that results from nerve damage in the periphery. The degeneration of sensory axon terminals leads to changes or loss of sensory functions, often manifesting as debilitating pain, weakness, numbness, tingling, and disability. The pathogenesis of most peripheral neuropathies remains to be fully elucidated. Cumulative evidence from both early and recent studies indicates that tubulin damage may provide a common underlying mechanism of axonal injury in various peripheral neuropathies. In particular, tubulin post-translational modifications have been recently implicated in both toxic and inherited forms of peripheral neuropathy through regulation of axonal transport and mitochondria dynamics. This knowledge forms a new area of investigation with the potential for developing therapeutic strategies to prevent or delay peripheral neuropathy by restoring tubulin homeostasis.

Protocol for observing microtubules and microtubule ends in both fixed and live primary microglia cells.

Microtubule dynamics and orientation have crucial roles in many vital cellular processes. However, functional live imaging of microtubules and/or microtubule ends in primary microglia can be challenging. Here, we present a protocol for observing microtubules and microtubule ends in both fixed and live primary microglia cells. We describe steps for microglia culture and in vitro stimulation, SiR-tubulin labeling, lentivirus preparation, live imaging, immunostaining, and image acquisition. We also provide procedures for SiR-tubulin, EB3-EGFP, and EB1 analyses. For complete details on the use and execution of this protocol, please refer to Rosito et al. (2023).1.

Pre-analytical and analytical variability of reticulocyte counts in dogs.

BACKGROUND: Automated fluorescence-based haematology analysers are now available for reticulocyte enumeration in veterinary medicine, but manual counting is still largely used. This study aimed to evaluate potential sources of analytical and pre-analytical errors when performing automated and manual counts. METHODS: Automated and two-operator double-blind manual reticulocyte counts were performed on 15 blood samples. The intra-assay variation of the automated and manual counts and the interoperator variation in the manual counts were then calculated. In addition, the effects of storage were evaluated using samples refrigerated at 4°C or stored at room temperature for 2, 4, 12, 24, 48 or 72 hours after sampling. RESULTS: Intra-assay coefficients of variation were lower for automated counts than for manual counts. Comparison between automated and mean total manual reticulocyte count showed no significant differences. In both refrigerated samples and those stored at room temperature, an increase in reticulocyte count was recorded only after 72 hours. Staining artefacts occurred only in one stored sample counted manually. LIMITATIONS: The presence of cytoplasmic particles other than RNA can cause misinterpretation of cells, leading to an erroneous reticulocyte count. CONCLUSION: The use of an automated analyser is preferable for reticulocyte enumeration in dogs. Common storage conditions seem to minimally affect reticulocyte evaluation; however, it is recommended to perform the analysis as soon as possible after sampling.

MFN2-dependent recruitment of ATAT1 coordinates mitochondria motility with alpha-tubulin acetylation and is disrupted in CMT2A.

Acetylated microtubules play key roles in the regulation of mitochondria dynamics. It has however remained unknown if the machinery controlling mitochondria dynamics functionally interacts with the alpha-tubulin acetylation cycle. Mitofusin-2 (MFN2), a large GTPase residing in the mitochondrial outer membrane and mutated in Charcot-Marie-Tooth type 2 disease (CMT2A), is a regulator of mitochondrial fusion, transport and tethering with the endoplasmic reticulum. The role of MFN2 in regulating mitochondrial transport has however remained elusive. Here we show that mitochondrial contacts with microtubules are sites of alpha-tubulin acetylation, which occurs through the MFN2-mediated recruitment of alpha-tubulin acetyltransferase 1 (ATAT1). We discover that this activity is critical for MFN2-dependent regulation of mitochondria transport, and that axonal degeneration caused by CMT2A MFN2 associated mutations, R94W and T105M, may depend on the inability to release ATAT1 at sites of mitochondrial contacts with microtubules. Our findings reveal a function for mitochondria in regulating acetylated alpha-tubulin and suggest that disruption of the tubulin acetylation cycle play a pathogenic role in the onset of MFN2-dependent CMT2A.

Microglia reactivity entails microtubule remodeling from acentrosomal to centrosomal arrays.

Microglia reactivity entails a large-scale remodeling of cellular geometry, but the behavior of the microtubule cytoskeleton during these changes remains unexplored. Here we show that activated microglia provide an example of microtubule reorganization from a non-centrosomal array of parallel and stable microtubules to a radial array of more dynamic microtubules. While in the homeostatic state, microglia nucleate microtubules at Golgi outposts, and activating signaling induces recruitment of nucleating material nearby the centrosome, a process inhibited by microtubule stabilization. Our results demonstrate that a hallmark of microglia reactivity is a striking remodeling of the microtubule cytoskeleton and suggest that while pericentrosomal microtubule nucleation may serve as a distinct marker of microglia activation, inhibition of microtubule dynamics may provide a different strategy to reduce microglia reactivity in inflammatory disease.

Alterations to the broad-spectrum formin inhibitor SMIFH2 modulate potency but not specificity.

SMIFH2 is a small molecule inhibitor of the formin family of cytoskeletal regulators that was originally identified in a screen for suppression of actin polymerization induced by the mouse formin Diaphanous 1 (mDia1). Despite widespread use of this compound, it is unknown whether SMIFH2 inhibits all human formins. Additionally, the nature of protein/inhibitor interactions remains elusive. We assayed SMIFH2 against human formins representing six of the seven mammalian classes and found inhibitory activity against all formins tested. We synthesized a panel of SMIFH2 derivatives and found that, while many alterations disrupt SMIFH2 activity, substitution of an electron-donating methoxy group in place of the bromine along with halogenation of the furan ring increases potency by approximately five-fold. Similar to SMIFH2, the active derivatives are also pan-inhibitors for the formins tested. This result suggests that while potency can be improved, the goal of distinguishing between highly conserved FH2 domains may not be achievable using the SMIFH2 scaffold.

Crosstalk between acetylation and the tyrosination/detyrosination cycle of α-tubulin in Alzheimer's disease.

Microtubules (MTs) support a variety of neuronal functions, such as maintenance of cell structure, transport, and synaptic plasticity. Neuronal MTs are highly heterogeneous due to several tubulin isotypes and the presence of multiple post-translational modifications, such as detyrosination and acetylation. The tubulin tyrosination/detyrosination cycle is a key player in the maintenance of MT dynamics, as tyrosinated tubulin is associated with more dynamic MTs, while detyrosinated tubulin is linked to longer lived, more stable MTs. Dysfunction of tubulin re-tyrosination was recently correlated to Alzheimer's disease progression. The implication of tubulin acetylation in Alzheimer's disease has, however, remained controversial. Here, we demonstrate that tubulin acetylation accumulates in post-mortem brain tissues from Alzheimer's disease patients and human neurons harboring the Alzheimer's familial APP-V717I mutation. We further show that tubulin re-tyrosination, which is defective in Alzheimer's disease, can control acetylated tubulin in primary neurons irrespective of the levels of the enzymes regulating tubulin acetylation, suggesting that reduced MT dynamics associated with impaired tubulin re-tyrosination might contribute to the accumulation of tubulin acetylation that we detected in Alzheimer's disease.

The microtubule cytoskeleton at the synapse.

In neurons, microtubules (MTs) provide routes for transport throughout the cell and structural support for dendrites and axons. Both stable and dynamic MTs are necessary for normal neuronal functions. Research in the last two decades has demonstrated that MTs play additional roles in synaptic structure and function in both pre- and postsynaptic elements. Here, we review current knowledge of the functions that MTs perform in excitatory and inhibitory synapses, as well as in the neuromuscular junction and other specialized synapses, and discuss the implications that this knowledge may have in neurological disease.

The synaptic life of microtubules.

In neurons, control of microtubule dynamics is required for multiple homeostatic and regulated activities. Over the past few decades, a great deal has been learned about the role of the microtubule cytoskeleton in axonal and dendritic transport, with a broad impact on neuronal health and disease. However, significantly less attention has been paid to the importance of microtubule dynamics in directly regulating synaptic function. Here, we review emerging literature demonstrating that microtubules enter synapses and control central aspects of synaptic activity, including neurotransmitter release and synaptic plasticity. The pleiotropic effects caused by a dysfunctional synaptic microtubule cytoskeleton may thus represent a key point of vulnerability for neurons and a primary driver of neurological disease.

Live imaging of microtubule dynamics at excitatory presynaptic boutons in primary hippocampal neurons and acute hippocampal slices.

Analyses of microtubule (MT) plus end dynamics at glutamatergic en passant boutons can be carried out in cultured primary neurons isolated from mouse or rat embryos or ex vivo in acute slices isolated from mice that had been electroporated in utero. Here, we describe a protocol for setting up and analyzing live image recordings of primary neurons and acute hippocampal slices expressing tagged versions of the MT plus end binding protein EB3 and the presynaptic vesicle markers vGlut1 or VAMP2. For complete information on the use and execution of this protocol, please refer to Qu et al. (2019).

Transthyretin Promotes Axon Growth via Regulation of Microtubule Dynamics and Tubulin Acetylation.

Transthyretin (TTR), a plasma and cerebrospinal fluid protein, increases axon growth and organelle transport in sensory neurons. While neurons extend their axons, the microtubule (MT) cytoskeleton is crucial for the segregation of functional compartments and axonal outgrowth. Herein, we investigated whether TTR promotes axon elongation by modulating MT dynamics. We found that TTR KO mice have an intrinsic increase in dynamic MTs and reduced levels of acetylated α-tubulin in peripheral axons. In addition, they failed to modulate MT dynamics in response to sciatic nerve injury, leading to decreased regenerative capacity. Importantly, restoring acetylated α-tubulin levels of TTR KO dorsal root ganglia (DRG) neurons using an HDAC6 inhibitor is sufficient to completely revert defective MT dynamics and neurite outgrowth. In summary, our results reveal a new role for TTR in the modulation of MT dynamics by regulating α-tubulin acetylation via modulation of the acetylase ATAT1, and suggest that this activity underlies TTR neuritogenic function.