Putting the brakes on axonal branching.

In a recent study, Ziak et al. employed precise sparse labeling and spatiotemporally controlled genetic manipulations to uncover novel regulators of axon branching of layer 2/3 mouse callosal projection neurons. The authors elucidated a cell-autonomous signaling pathway wherein glycogen synthase kinase 3ß (GSK3ß) phosphorylation of microtubule-associated protein 1B (MAP1B) restricts interstitial axon branching by modulating microtubule (MT) tyrosination status.

Efficient axonal transport of endolysosomes relies on the balanced ratio of microtubule tyrosination and detyrosination.

In neurons, the microtubule (MT) cytoskeleton forms the basis for long-distance protein transport from the cell body into and out of dendrites and axons. To maintain neuronal polarity, the axon initial segment (AIS) serves as a physical barrier, separating the axon from the somatodendritic compartment and acting as a filter for axonal cargo. Selective trafficking is further instructed by axonal enrichment of MT post-translational modifications, which affect MT dynamics and the activity of motor proteins. Here, we compared two knockout mouse lines lacking the respective enzymes for MT tyrosination and detyrosination, and found that both knockouts led to a shortening of the AIS. Neurons from both lines also showed an increased immobile fraction of endolysosomes present in the axon, whereas mobile organelles displayed shortened run distances in the retrograde direction. Overall, our results highlight the importance of maintaining the balance of tyrosinated and detyrosinated MTs for proper AIS length and axonal transport processes.

Detyrosinated microtubule arrays drive myofibrillar malformations in mdx muscle fibers.

Altered myofibrillar structure is a consequence of dystrophic pathology that impairs skeletal muscle contractile function and increases susceptibility to contraction injury. In murine Duchenne muscular dystrophy (mdx), myofibrillar alterations are abundant in advanced pathology (>4 months), an age where we formerly established densified microtubule (MT) arrays enriched in detyrosinated (deTyr) tubulin as negative disease modifiers impacting cell mechanics and mechanotransduction. Given the essential role of deTyr-enriched MT arrays in myofibrillar growth, maintenance, and repair, we examined the increased abundance of these arrays as a potential mechanism for these myofibrillar alterations. Here we find an increase in deTyr-tubulin as an early event in dystrophic pathology (4 weeks) with no evidence myofibrillar alterations. At 16 weeks, we show deTyr-enriched MT arrays significantly densified and co-localized to areas of myofibrillar malformation. Profiling the enzyme complexes responsible for deTyr-tubulin, we identify vasohibin 2 (VASH2) and small vasohibin binding protein (SVBP) significantly elevated in the mdx muscle at 4 weeks. Using the genetic increase in VASH2/SVBP expression in 4 weeks wild-type mice we find densified deTyr-enriched MT arrays that co-segregate with myofibrillar malformations similar to those in the 16 weeks mdx. Given that no changes in sarcomere organization were identified in fibers expressing sfGFP as a control, we conclude that disease-dependent densification of deTyr-enriched MT arrays underscores the altered myofibrillar structure in dystrophic skeletal muscle fibers.

The detyrosination/re-tyrosination cycle of tubulin and its role and dysfunction in neurons and cardiomyocytes.

Among the variety of post-translational modifications to which microtubules are subjected, the detyrosination/re-tyrosination cycle is specific to tubulin. It is conserved by evolution and characterized by the enzymatic removal and re-addition of a gene-encoded tyrosine residue at the C-terminus of α-tubulin. Detyrosinated tubulin can be further converted to Δ2-tubulin by the removal of an additional C-terminal glutamate residue. Detyrosinated and Δ2-tubulin are carried by stable microtubules whereas tyrosinated microtubules are present on dynamic polymers. The cycle regulates trafficking of many cargo transporting molecular motors and is linked to the microtubule dynamics via regulation of microtubule interactions with specific cellular effectors such as kinesin-13. Here, we give an historical overview of the general features discovered for the cycle. We highlight the recent progress toward structure and functioning of the enzymes that keep the levels of tyrosinated and detyrosinated tubulin in cells, the long-known tubulin tyrosine ligase and the recently discovered vasohibin-SVBP complexes. We further describe how the cycle controls microtubule functions in healthy neurons and cardiomyocytes and how deregulations of the cycle are involved in dysfunctions of these highly differentiated cells, leading to neurodegeneration and heart failure in humans.

VASH1-SVBP and VASH2-SVBP generate different detyrosination profiles on microtubules.

The detyrosination/tyrosination cycle of α-tubulin is critical for proper cell functioning. VASH1-SVBP and VASH2-SVBP are ubiquitous enzymes involved in microtubule detyrosination, whose mode of action is little known. Here, we show in reconstituted systems and cells that VASH1-SVBP and VASH2-SVBP drive the global and local detyrosination of microtubules, respectively. We solved the cryo-electron microscopy structure of VASH2-SVBP bound to microtubules, revealing a different microtubule-binding configuration of its central catalytic region compared to VASH1-SVBP. We show that the divergent mode of detyrosination between the two enzymes is correlated with the microtubule-binding properties of their disordered N- and C-terminal regions. Specifically, the N-terminal region is responsible for a significantly longer residence time of VASH2-SVBP on microtubules compared to VASH1-SVBP. We suggest that this VASH region is critical for microtubule detachment and diffusion of VASH-SVBP enzymes on lattices. Our results suggest a mechanism by which VASH1-SVBP and VASH2-SVBP could generate distinct microtubule subpopulations and confined areas of detyrosinated lattices to drive various microtubule-based cellular functions.

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.

Posttranslational modification of microtubules by the MATCAP detyrosinase.

The detyrosination-tyrosination cycle involves the removal and religation of the C-terminal tyrosine of α-tubulin and is implicated in cognitive, cardiac, and mitotic defects. The vasohibin-small vasohibin-binding protein (SVBP) complex underlies much, but not all, detyrosination. We used haploid genetic screens to identify an unannotated protein, microtubule associated tyrosine carboxypeptidase (MATCAP), as a remaining detyrosinating enzyme. X-ray crystallography and cryo-electron microscopy structures established MATCAP's cleaving mechanism, substrate specificity, and microtubule recognition. Paradoxically, whereas abrogation of tyrosine religation is lethal in mice, codeletion of MATCAP and SVBP is not. Although viable, defective detyrosination caused microcephaly, associated with proliferative defects during neurogenesis, and abnormal behavior. Thus, MATCAP is a missing component of the detyrosination-tyrosination cycle, revealing the importance of this modification in brain formation.

Tubulin Carboxypeptidase Activity Promotes Focal Gelatin Degradation in Breast Tumor Cells and Induces Apoptosis in Breast Epithelial Cells That Is Overcome by Oncogenic Signaling.

Post-translational modifications (PTMs) of the microtubule network impart differential functions across normal cell types and their cancerous counterparts. The removal of the C-terminal tyrosine of α-tubulin (deTyr-Tub) as performed by the tubulin carboxypeptidase (TCP) is of particular interest in breast epithelial and breast cancer cells. The recent discovery of the genetic identity of the TCP to be a vasohibin (VASH1/2) coupled with a small vasohibin-binding protein (SVBP) allows for the functional effect of this tubulin PTM to be directly tested for the first time. Our studies revealed the immortalized breast epithelial cell line MCF10A undergoes apoptosis following transfection with TCP constructs, but the addition of oncogenic KRas or Bcl-2/Bcl-xL overexpression prevents subsequent apoptotic induction in the MCF10A background. Functionally, an increase in deTyr-Tub via TCP transfection in MDA-MB-231 and Hs578t breast cancer cells leads to enhanced focal gelatin degradation. Given the elevated deTyr-Tub at invasive tumor fronts and the correlation with poor breast cancer survival, these new discoveries help clarify how the TCP synergizes with oncogene activation, increases focal gelatin degradation, and may correspond to increased tumor cell invasion. These connections could inform more specific microtubule-directed therapies to target deTyr-tubulin.

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.

Oleoylethanolamide Delays the Dysfunction and Death of Purkinje Cells and Ameliorates Behavioral Defects in a Mouse Model of Cerebellar Neurodegeneration.

Oleoylethanolamide (OEA) is an endocannabinoid that has been proposed to prevent neuronal damage and neuroinflammation. In this study, we evaluated the effects of OEA on the disruption of both cerebellar structure and physiology and on the behavior of Purkinje cell degeneration (PCD) mutant mice. These mice exhibit cerebellar degeneration, displaying microtubule alterations that trigger the selective loss of Purkinje cells and consequent behavioral impairments. The effects of different doses (1, 5, and 10 mg/kg, i.p.) and administration schedules (chronic and acute) of OEA were assessed at the behavioral, histological, cellular, and molecular levels to determine the most effective OEA treatment regimen. Our in vivo results demonstrated that OEA treatment prior to the onset of the preneurodegenerative phase prevented morphological alterations in Purkinje neurons (the somata and dendritic arbors) and decreased Purkinje cell death. This effect followed an inverted U-shaped time-response curve, with acute administration on postnatal day 12 (10 mg/kg, i.p.) being the most effective treatment regimen tested. Indeed, PCD mice that received this specific OEA treatment regimen showed improvements in motor, cognitive and social functions, which were impaired in these mice. Moreover, these in vivo neuroprotective effects of OEA were mediated by the PPARα receptor, as pretreatment with the PPARα antagonist GW6471 (2.5 mg/kg, i.p.) abolished them. Finally, our in vitro results suggested that the molecular effect of OEA was related to microtubule stability and structure since OEA administration normalized some alterations in microtubule features in PCD-like cells. These findings provide strong evidence supporting the use of OEA as a pharmacological agent to limit severe cerebellar neurodegenerative processes.

Proteomic and Functional Studies Reveal Detyrosinated Tubulin as Treatment Target in Sarcomere Mutation-Induced Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. While ≈50% of patients with HCM carry a sarcomere gene mutation (sarcomere mutation-positive, HCMSMP), the genetic background is unknown in the other half of the patients (sarcomere mutation-negative, HCMSMN). Genotype-specific differences have been reported in cardiac function. Moreover, HCMSMN patients have later disease onset and a better prognosis than HCMSMP patients. To define if genotype-specific derailments at the protein level may explain the heterogeneity in disease development, we performed a proteomic analysis in cardiac tissue from a clinically well-phenotyped HCM patient group. METHODS: A proteomics screen was performed in cardiac tissue from 39 HCMSMP patients, 11HCMSMN patients, and 8 nonfailing controls. Patients with HCM had obstructive cardiomyopathy with left ventricular outflow tract obstruction and diastolic dysfunction. A novel MYBPC32373insG mouse model was used to confirm functional relevance of our proteomic findings. RESULTS: In all HCM patient samples, we found lower levels of metabolic pathway proteins and higher levels of extracellular matrix proteins. Levels of total and detyrosinated α-tubulin were markedly higher in HCMSMP than in HCMSMN and controls. Higher tubulin detyrosination was also found in 2 unrelated MYBPC3 mouse models and its inhibition with parthenolide normalized contraction and relaxation time of isolated cardiomyocytes. CONCLUSIONS: Our findings indicate that microtubules and especially its detyrosination contribute to the pathomechanism of patients with HCMSMP. This is of clinical importance since it represents a potential treatment target to improve cardiac function in patients with HCMSMP, whereas a beneficial effect may be limited in patients with HCMSMN.

Tubulin post-translational modifications control neuronal development and functions.

Microtubules (MTs) are an essential component of the neuronal cytoskeleton; they are involved in various aspects of neuron development, maintenance, and functions including polarization, synaptic plasticity, and transport. Neuronal MTs are highly heterogeneous due to the presence of multiple tubulin isotypes and extensive post-translational modifications (PTMs). These PTMs-most notably detyrosination, acetylation, and polyglutamylation-have emerged as important regulators of the neuronal microtubule cytoskeleton. With this review, we summarize what is currently known about the impact of tubulin PTMs on microtubule dynamics, neuronal differentiation, plasticity, and transport as well as on brain function in normal and pathological conditions, in particular during neuro-degeneration. The main therapeutic approaches to neuro-diseases based on the modulation of tubulin PTMs are also summarized. Overall, the review indicates how tubulin PTMs can generate a large number of functionally specialized microtubule sub-networks, each of which is crucial to specific neuronal features.

Depletion of Vasohibin 1 Speeds Contraction and Relaxation in Failing Human Cardiomyocytes.

RATIONALE: Impaired myocardial relaxation is an intractable feature of several heart failure (HF) causes. In human HF, detyrosinated microtubules stiffen cardiomyocytes and impair relaxation. Yet the identity of detyrosinating enzymes have remained ambiguous, hindering mechanistic study and therapeutic development. OBJECTIVE: We aimed to determine if the recently identified complex of VASH1/2 (vasohibin 1/2) and SVBP (small vasohibin binding protein) is an active detyrosinase in cardiomyocytes and if genetic inhibition of VASH-SVBP is sufficient to lower stiffness and improve contractility in HF. METHODS AND RESULTS: Transcriptional profiling revealed that VASH1 transcript is >10-fold more abundant than VASH2 in human hearts. Using short hairpin RNAs (shRNAs) against VASH1, VASH2, and SVBP, we showed that both VASH1- and VASH2-SVBP complexes function as tubulin carboxypeptidases in cardiomyocytes, with a predominant role for VASH1. We also generated a catalytically dead version of the tyrosinating enzyme TTL (TTL-E331Q) to separate the microtubule depolymerizing effects of TTL from its enzymatic activity. Assays of microtubule stability revealed that both TTL and TTL-E331Q depolymerize microtubules, while VASH1 and SVBP depletion reduce detyrosination independent of depolymerization. We next probed effects on human cardiomyocyte contractility. Contractile kinetics were slowed in HF, with dramatically slowed relaxation in cardiomyocytes from patients with HF with preserved ejection fraction. Knockdown of VASH1 conferred subtle kinetic improvements in nonfailing cardiomyocytes, while markedly improving kinetics in failing cardiomyocytes. Further, TTL, but not TTL-E331Q, robustly sped relaxation. Simultaneous measurements of calcium transients and contractility demonstrated that VASH1 depletion speeds kinetics independent from alterations to calcium cycling. Finally, atomic force microscopy confirmed that VASH1 depletion reduces the stiffness of failing human cardiomyocytes. CONCLUSIONS: VASH-SVBP complexes are active tubulin carboxypeptidases in cardiomyocytes. Inhibition of VASH1 or activation of TTL is sufficient to lower stiffness and speed relaxation in cardiomyocytes from patients with HF, supporting further pursuit of detyrosination as a therapeutic target for diastolic dysfunction.

Defective tubulin detyrosination causes structural brain abnormalities with cognitive deficiency in humans and mice.

Reversible detyrosination of tubulin, the building block of microtubules, is crucial for neuronal physiology. Enzymes responsible for detyrosination were recently identified as complexes of vasohibins (VASHs) one or two with small VASH-binding protein (SVBP). Here we report three consanguineous families, each containing multiple individuals with biallelic inactivation of SVBP caused by truncating variants (p.Q28* and p.K13Nfs*18). Affected individuals show brain abnormalities with microcephaly, intellectual disability and delayed gross motor and speech development. Immunoblot testing in cells with pathogenic SVBP variants demonstrated that the encoded proteins were unstable and non-functional, resulting in a complete loss of VASH detyrosination activity. Svbp knockout mice exhibit drastic accumulation of tyrosinated tubulin and a reduction of detyrosinated tubulin in brain tissue. Similar alterations in tubulin tyrosination levels were observed in cultured neurons and associated with defects in axonal differentiation and architecture. Morphological analysis of the Svbp knockout mouse brains by anatomical magnetic resonance imaging showed a broad impact of SVBP loss, with a 7% brain volume decrease, numerous structural defects and a 30% reduction of some white matter tracts. Svbp knockout mice display behavioural defects, including mild hyperactivity, lower anxiety and impaired social behaviour. They do not, however, show prominent memory defects. Thus, SVBP-deficient mice recapitulate several features observed in human patients. Altogether, our data demonstrate that deleterious variants in SVBP cause this neurodevelopmental pathology, by leading to a major change in brain tubulin tyrosination and alteration of microtubule dynamics and neuron physiology.

Structural basis of tubulin detyrosination by the vasohibin-SVBP enzyme complex.

Vasohibins are tubulin tyrosine carboxypeptidases that are important in neuron physiology. We examined the crystal structures of human vasohibin 1 and 2 in complex with small vasohibin-binding protein (SVBP) in the absence and presence of different inhibitors and a C-terminal α-tubulin peptide. In combination with functional data, we propose that SVBP acts as an activator of vasohibins. An extended groove and a distinctive surface residue patch of vasohibins define the specific determinants for recognizing and cleaving the C-terminal tyrosine of α-tubulin and for binding microtubules, respectively. The vasohibin-SVBP interaction and the ability of the enzyme complex to associate with microtubules regulate axon specification of neurons. Our results define the structural basis of tubulin detyrosination by vasohibins and show the relevance of this process for neuronal development. Our findings offer a unique platform for developing drugs against human conditions with abnormal tubulin tyrosination levels, such as cancer, heart defects and possibly brain disorders.

Author Correction: Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Cytoskeleton stability is essential for the integrity of the cerebellum and its motor- and affective-related behaviors.

The cerebellum plays a key role in motor tasks, but its involvement in cognition is still being considered. Although there is an association of different psychiatric and cognitive disorders with cerebellar impairments, the lack of time-course studies has hindered the understanding of the involvement of cerebellum in cognitive and non-motor functions. Such association was here studied using the Purkinje Cell Degeneration mutant mouse, a model of selective and progressive cerebellar degeneration that lacks the cytosolic carboxypeptidase 1 (CCP1). The effects of the absence of this enzyme on the cerebellum of mutant mice were analyzed both in vitro and in vivo. These analyses were carried out longitudinally (throughout both the pre-neurodegenerative and neurodegenerative stages) and different motor and non-motor tests were performed. We demonstrate that the lack of CCP1 affects microtubule dynamics and flexibility, defects that contribute to the morphological alterations of the Purkinje cells (PCs), and to progressive cerebellar breakdown. Moreover, this degeneration led not only to motor defects but also to gradual cognitive impairments, directly related to the progression of cellular damage. Our findings confirm the cerebellar implication in non-motor tasks, where the formation of the healthy, typical PCs structure is necessary for normal cognitive and affective behavior.

Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing.

In this report, we present an improved protocol for CRISPR/Cas9 genome editing in mice. The procedure consists in the electroporation of intact mouse zygotes with ribonucleoprotein complexes prepared in vitro from recombinant Cas9 nuclease and synthetic dual guide RNA. This simple cloning-free method proves to be extremely efficient for the generation of indels and small deletions by non-homologous end joining, and for the generation of specific point mutations by homology-directed repair. The procedure, which avoids DNA construction, in vitro transcription and oocyte microinjection, greatly simplifies genome editing in mice.

Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation.

Reversible detyrosination of α-tubulin is crucial to microtubule dynamics and functions, and defects have been implicated in cancer, brain disorganization, and cardiomyopathies. The identity of the tubulin tyrosine carboxypeptidase (TCP) responsible for detyrosination has remained unclear. We used chemical proteomics with a potent irreversible inhibitor to show that the major brain TCP is a complex of vasohibin-1 (VASH1) with the small vasohibin binding protein (SVBP). VASH1 and its homolog VASH2, when complexed with SVBP, exhibited robust and specific Tyr/Phe carboxypeptidase activity on microtubules. Knockdown of vasohibins or SVBP and/or inhibitor addition in cultured neurons reduced detyrosinated α-tubulin levels and caused severe differentiation defects. Furthermore, knockdown of vasohibins disrupted neuronal migration in developing mouse neocortex. Thus, vasohibin/SVBP complexes represent long-sought TCP enzymes.