Biology subjects

Moutin, M.-J.

Publications and source records attributed to Moutin, M.-J..

9 recordsLinked to original sources

bioRxiv2026

VASH1 phosphorylation regulates tubulin detyrosination via CNP/PRKG1α signaling in DRG neurons.

Functional neuronal circuit formation requires precise regulation of axon growth and branching through dynamic cytoskeletal remodeling. Although microtubule detyrosination/re-tyrosination critically controls microtubule dynamics, how extracellular signals regulate this process remains unclear. Here, we identify a direct link between C-type natriuretic peptide, protein kinase cGMP-dependent 1 (CNP/cGMP/PRKG1) signaling and microtubule detyrosination in dorsal root ganglion (DRG) neurons. CNP stimulation induces growth cone enlargement, increases tyrosinated tubulin within growth cones, and reduces detyrosinated tubulin along axons, effects abolished by PRKG1 inhibition. Pharmacological inhibition or genetic deletion of the detyrosinating enzyme VASH1 phenocopies CNP-induced growth cone remodeling. In vitro kinase assays identify VASH1 as a direct PRKG1 substrate and reveal a specific C-terminal serine phosphorylation site (serine 337) that suppresses VASH1-SVBP microtubule association and detyrosination activity. Together, these findings establish VASH1-SVBP as a key downstream effector linking extracellular guidance signaling to microtubule remodeling during DRG neuronal development.

cell biology↗

bioRxiv2026

Neprilysin inhibition reduces microtubule detyrosination in cardiomyocytes through a cGMP-PRKG1-VASH1 axis

Microtubule detyrosination and re-tyrosination on the C-terminus of -tubulin are mediated by the vasohibin (VASH)-small vasohibin-binding protein (SVBP) complex and tubulin tyrosine ligase (TTL), respectively. Elevated levels of detyrosinated -tubulin (dTyr-tub) are observed in heart failure, and reducing this modification improves cardiac function, suggesting that clinically used heart failure therapies may modulate microtubule detyrosination. We investigated whether sacubitrilat and valsartan, the active components of the angiotensin receptor-neprilysin inhibitor LCZ696, influence dTyr-tub levels in endothelin-1 (ET1)-induced hypertrophy in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). While both sacubitrilat and valsartan prevented hypertrophy, only sacubitrilat prevented ET1-induced dTyr-tub accumulation. RNA sequencing revealed that sacubitrilat normalized several ET1-induced dysregulated pathways. Sacubitrilat slightly increased cyclic guanosine 3,5-monophosphate (cGMP) levels and lowered dTyr-tub, whereas inhibition or knockdown of the cGMP-dependent protein kinase 1 (PRKG1) increased dTyr-tub level. Mechanistically, PRKG1 alpha phosphorylated native VASH1. Incubation of microtubules with the VASH1-SVBP complex containing wild-type VASH1 increased detyrosination, while incubation of the complex containing a VASH1 phosphomimic, in which seven C-terminal serine residues were mutated to glutamate (VASH1-7E) did not. Consistently, overexpression of VASH1-7E gave rise to lower dTyr-tub level than overexpression of a non-phosphorylatable form of VASH1 (VASH1-7A) in hiPSC-CMs deficient in VASH1. In conclusion, these findings identify a cGMP-PRKG1-VASH1 signaling axis that reduces microtubule detyrosination in cardiomyocytes. Our work provides mechanistic insight into how neprilysin inhibition may contribute to therapeutic benefit in heart failure. One Sentence SummaryWe establish a neprilysin-cGMP-PRKG1-VASH1 signaling axis that reduces microtubule detyrosination in cardiomyocytes.

cell biology↗

bioRxiv2026

Phosphoproteomics of Hypertrophic Cardiomyopathy Patient Myocardium and Novel hiPSC-CM Model Reveal Protein Kinase A as a Modulator of Microtubule Repolymerization

Background and aimsIncreased levels of -tubulin and its post-translational modifications (PTMs) are found in human heart failure and could initiate diastolic dysfunction by modulating cardiomyocyte stiffness. How these modifications occur and how they may underlie cardiac dysfunction remains unknown. Upstream kinases may play a critical role, but this has not been explored. Methods and resultsHere we address this question by, for the first time ever, determining levels of the enzymes involved in microtubule (MT) detyrosination and acetylation (TAT1, HDAC6) in a well-characterized cohort of patients with hypertrophic cardiomyopathy (HCM). In HCM patients (N=10-11), protein levels of detyrosination enzymes remain unaltered, whilst levels of TAT1 and HDAC6 were decreased and increased, respectively. Phosphoproteomics in HCM (N=24) and control (N=8) myocardium identified significant differences in over 1900 serine/threonine and 160 tyrosine phosphosites, in addition to increased EGFR/IGF1R-MAPK signaling in HCM. We subsequently showed that MT repolymerization was increased in HCM MYBPC3Arg943X hiPSC-CMs. Isoprenaline-mediated PKA activation decreased MT repolymerization in hiPSC-CMs and revealed CLASP1, MAST4 and MAP1A as potential MT modifiers in HCM. ConclusionsWe show that the altered HCM MT code cannot be attributed to levels of key MT-modifying enzymes. By combining kinome analyses in human HCM hearts with hiPSC-CM studies on MT dynamics, PTMs and contractility we unveiled a regulatory role for MTs in the cardiomyocyte response to beta-adrenergic receptor stimulation. Disease-mediated changes in the MT code thereby exert both a direct, and indirect effect on cardiac function via mediating the response to adrenergic activation. Graphical Abstract created with BioRender.com O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/706710v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1c58dc4org.highwire.dtl.DTLVardef@de502eorg.highwire.dtl.DTLVardef@1621512org.highwire.dtl.DTLVardef@557b82_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2026

SHANK3-CAMSAP2 interaction links synapses to dendritic microtubule organisation in PV neurons

The microtubule cytoskeleton plays an essential role in establishing and maintaining neuronal polarity. In neurons, microtubules are initially generated at the centrosome which gradually loses this role in development. In mature neurons, microtubules are stabilised by the microtubule minus end-binding protein CAMSAP2. How and where microtubule minus ends are anchored within dendrites of mammalian neurons remains an open question. Here, we show that microtubules are directly attached to the postsynaptic density of excitatory synapses through an interaction of CAMSAP2 with the scaffolding protein SHANK3. This process is particularly relevant in parvalbumin-positive GABAergic neurons, in which excitatory synapses are predominantly located directly on the dendritic shaft in direct proximity to microtubules. Notably, this association is strongly enhanced by the Autism Spectrum Disorder (ASD)-associated SHANK3L68P mutation, leading to an increase of synaptic levels of CAMSAP2. This promotes an increased microtubule association with the synapse and alters microtubule dynamics. Downregulation of CAMSAP2 in SHANK3L68P parvalbumin neurons tips the balance between site-specific microtubule stabilisation and dynamics, reshaping dendritic architecture, connecting SHANK3-CAMSAP2 dependent microtubule regulation to synaptic ASD pathology. TeaserInteraction between synapses and microtubules is enhanced in parvalbumin neurons bearing the ASD-associated SHANK3 L68P mutation.

neuroscience↗

bioRxiv2024

L-Dopa incorporation into tubulin alters microtubule dynamics and reduces dendritic spine invasion and synapse maintenance

Previous studies have shown that L-Dopa, a tyrosine analog used in Parkinsons disease treatment, can be incorporated into -tubulin C-terminal tail via the tubulin tyrosine ligase (TTL) and polymerize into microtubules. In this work, we demonstrated that mature wild type hippocampal neurons treated with L-Dopa exhibited reduced dendritic spine density, primarily affecting mature dendritic spines. In these neurons, L-Dopa treatment significantly reduced tyrosinated -tubulin levels without altering detyrosinated or {Delta}2 -tubulin levels, suggesting the formation of a new tubulin pool, likely composed of L-Dopa--tubulin. In vitro analysis of the activity of the purified VASH1-SVBP complex, the most abundant tubulin carboxypeptidase in brain, revealed that L-Dopa incorporation into -tubulin modified the binding of the complex to microtubules and reduced its carboxypeptidase activity. These results suggest that L-Dopa incorporation into tubulin alters the properties of microtubules and affects their ability to interact with the enzyme. To confirm the implication of L-Dopa-microtubules in dendritic spine alterations observed in wild type neurons, we analyzed the effect of L-Dopa treatment in neurons lacking the enzymes of the -tubulin detyrosination/tyrosination cycle. In these cells, L-Dopa cannot be incorporated into -tubulin due to the absence of the ligase (in TTL KO neurons) or the reduction of detyrosinated -tubulin levels (in SVBP KO neurons). L-Dopa treatment did not modify dendritic spine density in TLL KO or SVBP KO neurons, clearly demonstrating that the alterations in dendritic spines seen in WT neurons are due to the incorporation of L-Dopa into tubulin. Further analysis revealed that L-Dopa treatment decreased the percentage of spines containing excitatory synapses in wild type neurons, but not in TTL KO or SVBP KO neurons, suggesting a cumulative synaptic defect due to L-Dopa incorporation into microtubules. Additionally, L-Dopa altered microtubule dynamics by increasing catastrophe frequency and reducing comet lifetime, which led to fewer microtubules entering dendritic spines and decreased spine resistance to pruning. Taken together, our results demonstrate that L-Dopa incorporation into -tubulin drastically affects synaptic homeostasis, reaffirming the importance of balanced detyrosination/tyrosination of tubulin within the synaptic compartment. The abnormal dynamics of L-Dopa-microtubules and the reduction of dendritic spines and excitatory synapses highlight a novel mechanism of L-Dopa-induced synaptotoxicity.

neuroscience↗

bioRxiv2023

Efficient axonal trafficking of endolysosomes depends 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 axon. 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 we 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/detyrosinated MT for proper AIS length and axonal transport processes. Summary StatementDespite opposite effects on microtubule dynamics, shifting the balance of microtubule tyrosination/detyrosination in either direction resulted in surprisingly similar defects in axonal organelle transport.

neuroscience↗

bioRxiv2023

Myofibrillar malformations that arise in mdx muscle fibers are driven by detyrosinated microtubules

In Duchenne muscular dystrophy (DMD), alterations in the myofibrillar structure of skeletal muscle fibers that impair contractile function and increase injury susceptibility arise as a consequence of dystrophic pathology. In murine DMD (mdx), myofibrillar alterations are abundant in advanced pathology (>4 months), an age where we formerly established the densification of microtubules (MTs) post-translationally modified by detyrosination (deTyr-MTs) as a negative disease modifier. Given the essential role of MTs in myofibrillar growth, maintenance, and repair, we examined the increased abundance of deTyr-MTs as a potential mechanism for these myofibrillar alterations. Here we find increased levels of deTyr-MTs as an early event in dystrophic pathology (4 weeks) with no evidence of myofibrillar alterations. At 16 weeks, we show the level of deTyr-MTs is significantly increased 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 wks. We then use the genetic increase in VASH2/SVBP expression in 4 wk wild-type mice and find densified deTyr-MTs that co-segregate with myofibrillar malformations similar to those in the 16 wk mdx. Given that no changes were identified in fibers expressing EGFP as a control, we conclude that disease dependent densification of deTyr-MTs underscores the altered myofibrillar structure in dystrophic skeletal muscle fibers.

pathology↗

bioRxiv2022

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

The detyrosination/tyrosination cycle of -tubulin is critical for proper cell functioning. VASH1-SVBP and VASH2-SVBP are ubiquitous enzyme complexes involved in microtubule detyrosination. However, little is known about their mode of action. Here, we show in reconstituted systems and in cells that VASH1-SVBP and VASH2-SVBP drive global and local detyrosination of microtubules, respectively. We solved the cryo-electron microscopy structure of human VASH2-SVBP bound to microtubules, revealing a different microtubule-binding configuration of its central catalytic region compared to VASH1-SVBP. We further 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 domain is critical for microtubule-detachment and diffusion of VASH-SVBP enzymes on the lattice. Together, our results suggest a mechanism by which these enzymes could generate distinct microtubule subpopulations and confined areas of detyrosinated lattices to drive various microtubule-based cellular functions. SUMMARYVASH1-SVBP and VASH2-SVBP produce global and local detyrosination patterns of microtubule lattices, respectively. These activities rely on the interplay between the N- and C-terminal disordered regions of the enzymes, which determine their differential molecular mechanism of action. GRAPHICAL ABSTRACTSchematic representation of divergent molecular mechanisms of action of VASH-SVBP detyrosination complexes.

cell biology↗

bioRxiv2021

Impaired α-tubulin re-tyrosination leads to synaptic dysfunction and is a feature of Alzheimer's disease

In neurons, dynamic microtubules play regulatory roles in neurotransmission and synaptic plasticity. While stable microtubules contain detyrosinated tubulin, dynamic microtubules are composed of tyrosinated tubulin, suggesting that the tubulin tyrosination/detyrosination (Tyr/deTyr) cycle modulates microtubule dynamics and synaptic function. In the Tyr/deTyr cycle, the C-terminal tyrosine of -tubulin is re-added by tubulin-tyrosine-ligase (TTL). Here we show that TTL+/- mice exhibit decreased tyrosinated microtubules, synaptic plasticity and memory deficits, and that reduced TTL expression is a feature of sporadic and familial Alzheimers disease (AD), with human APPV717I neurons having less dynamic microtubules. We find that spines visited by dynamic microtubules are more resistant to Amyloid{beta}1-42 and that TTL, by promoting microtubule entry into spines, prevents A{beta}1-42-induced spine pruning. Our results demonstrate that the Tyr/deTyr cycle regulates synaptic plasticity, is protective against spine injury, and that tubulin re-tyrosination is lost in AD, providing evidence that a defective Tyr/deTyr cycle may contribute to neurodegeneration.

neuroscience↗