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.

Interplay between Triadin and Calsequestrin in the Pathogenesis of CPVT in the Mouse.

Recessive forms of catecholaminergic polymorphic ventricular tachycardia (CPVT) are induced by mutations in genes encoding triadin or calsequestrin, two proteins that belong to the Ca2+ release complex, responsible for intracellular Ca2+ release triggering cardiac contractions. To better understand the mechanisms of triadin-induced CPVT and to assay multiple therapeutic interventions, we used a triadin knockout mouse model presenting a CPVT-like phenotype associated with a decrease in calsequestrin protein level. We assessed different approaches to rescue protein expression and to correct intracellular Ca2+ release and cardiac function: pharmacological treatment with kifunensine or a viral gene transfer-based approach, using adeno-associated virus serotype 2/9 (AAV2/9) encoding the triadin or calsequestrin. We observed that the levels of triadin and calsequestrin are intimately linked, and that reduction of both proteins contributes to the CPVT phenotype. Different combinations of triadin and calsequestrin expression level were obtained using these therapeutic approaches. A full expression of each is not necessary to correct the phenotype; a fine-tuning of the relative re-expression of both triadin and calsequestrin is required to correct the CPVT phenotype and rescue the cardiac function. AAV-mediated gene delivery of calsequestrin or triadin and treatment with kifunensine are potential treatments for recessive forms of CPVT due to triadin mutations.

MAP6 interacts with Tctex1 and Cav 2.2/N-type calcium channels to regulate calcium signalling in neurons.

MAP6 proteins were first described as microtubule-stabilizing agents, whose properties were thought to be essential for neuronal development and maintenance of complex neuronal networks. However, deletion of all MAP6 isoforms in MAP6 KO mice does not lead to dramatic morphological aberrations of the brain but rather to alterations in multiple neurotransmissions and severe behavioural impairments. A search for protein partners of MAP6 proteins identified Tctex1 - a dynein light chain with multiple non-microtubule-related functions. The involvement of Tctex1 in calcium signalling led to investigate it in MAP6 KO neurons. In this study, we show that functional Cav 2.2/N-type calcium channels are deficient in MAP6 KO neurons, due to improper location. We also show that MAP6 proteins interact directly with both Tctex1 and the C-terminus of Cav 2.2/N-type calcium channels. A balance of these two interactions seems to be crucial for MAP6 to modulate calcium signalling in neurons.

Ataxia with cerebellar lesions in mice expressing chimeric PrP-Dpl protein.

Mutations within the central region of prion protein (PrP) have been shown to be associated with severe neurotoxic activity similar to that observed with Dpl, a PrP-like protein. To further investigate this neurotoxic effect, we generated lines of transgenic (Tg) mice expressing three different chimeric PrP-Dpl proteins. Chi1 (amino acids 1-57 of Dpl replaced by amino acids 1-125 of PrP) and Chi2 (amino acids 1-66 of Dpl replaced by amino acids 1-134 of PrP) abrogated the pathogenicity of Dpl indicating that the presence of a N-terminal domain of PrP (23-134) reduced the toxicity of Dpl, as reported. However, when the amino acids 1-24 of Dpl were replaced by amino acids 1-124 of PrP, Chi3 Tg mice, which express the chimeric protein at a very low level, start developing ataxia at the age of 5-7 weeks. This phenotype was not counteracted by a single copy of full-length-PrP(c) but rather by its overexpression, indicating the strong toxicity of the chimeric protein Chi3. Chi3 Tg mice exhibit severe cerebellar atrophy with a significant loss of granule cells. We concluded that aa25 to aa57 of Dpl, which are not present in Chi1 and Chi2 constructs, confer toxicity to the protein. We tested this possibility by using the 25-57 Dpl peptide in primary culture of mouse embryo cortical neurons and found a significant neurotoxic effect. This finding identifies a protein domain that plays a role in mediating Dpl-related toxicity.

Impact of manganese on primary hippocampal neurons from rodents.

Manganese-enhanced magnetic resonance imaging (MEMRI) is a powerful tool for in vivo tract tracing or functional imaging of the central nervous system. However Mn(2+) may be toxic at high levels. In this study, we addressed the impact of Mn(2+) on mouse hippocampal neurons (HN) and neuron-like N2a cells in culture, using several approaches. Both HN and N2a cells not exposed to exogenous MnCl2 were shown by synchrotron X-ray fluorescence to contain 5 mg/g Mn. Concentrations of Mn(2+) leading to 50% lethality (LC50) after 24 h of incubation were much higher for N2a cells (863 mM) than for HN (90 mM). The distribution of Mn(2+) in both cell types exposed to Mn(2+) concentrations below LC50 was perinuclear whereas that in cells exposed to concentrations above LC50 was more diffuse, suggesting an overloading of cell storage/detoxification capacity. In addition, Mn(2+) had a cell-type and dose-dependent impact on the total amount of intracellular P, Ca, Fe and Zn measured by synchrotron X-ray fluorescence. For HN neurons, immunofluorescence studies revealed that concentrations of Mn(2+) below LC50 shortened neuritic length and decreased mitochondria velocity after 24 h of incubation. Similar concentrations of Mn(2+) also facilitated the opening of the mitochondrial permeability transition pore in isolated mitochondria from rat brains. The sensitivity of primary HN to Mn(2+) demonstrated here supports their use as a relevant model to study Mn(2+) -induced neurotoxicity.

Nanoscale surface topography reshapes neuronal growth in culture.

Neurons are sensitive to topographical cues provided either by in vivo or in vitro environments on the micrometric scale. We have explored the role of randomly distributed silicon nanopillars on primary hippocampal neurite elongation and axonal differentiation. We observed that neurons adhere on the upper part of nanopillars with a typical distance between adhesion points of about 500 nm. These neurons produce fewer neurites, elongate faster, and differentiate an axon earlier than those grown on flat silicon surfaces. Moreover, when confronted with a differential surface topography, neurons specify an axon preferentially on nanopillars. As a whole, these results highlight the influence of the physical environment in many aspects of neuronal growth.

Quantitative Image Analysis of Axonal Morphology in In Vivo Model.

Quantifying axonal branching is crucial for understanding neural circuit function, developmental and regeneration processes and disease mechanisms. Factors that regulate patterns of axonal arborization and tune neuronal circuits are investigated for their implication in various disorders in brain connectivity. The lack of a reliable and user-friendly method makes the quantitative analysis of axon morphology difficult. Specifically, methods to visualize and quantify the complex axon arborization are challenging to implement and apply practically. Our study was aimed at developing a robust but simple method of quantification that used ImageJ 2D analysis and compared it with Imaris visualization and analysis of 3D images. We used zebrafish fluorescent transgenic lines to perform in vivo imaging of developing motor neuron axons that adequately reflected the complexity of axonal networks. Our new method, developed on ImageJ, is easy and fast, giving access to new information such as collateral distribution along the axonal shaft. This study describes step-by-step procedures that can be easily applied to a variety of organisms and in vitro systems. Our study provides a basis for further exploration of neural circuits to gain new insights into neuronal disorders and potential therapeutic interventions.

Huntingtin recruits KIF1A to transport synaptic vesicle precursors along the mouse axon to support synaptic transmission and motor skill learning.

Neurotransmitters are released at synapses by synaptic vesicles (SVs), which originate from SV precursors (SVPs) that have traveled along the axon. Because each synapse maintains a pool of SVs, only a small fraction of which are released, it has been thought that axonal transport of SVPs does not affect synaptic function. Here, studying the corticostriatal network both in microfluidic devices and in mice, we find that phosphorylation of the Huntingtin protein (HTT) increases axonal transport of SVPs and synaptic glutamate release by recruiting the kinesin motor KIF1A. In mice, constitutive HTT phosphorylation causes SV over-accumulation at synapses, increases the probability of SV release, and impairs motor skill learning on the rotating rod. Silencing KIF1A in these mice restored SV transport and motor skill learning to wild-type levels. Axonal SVP transport within the corticostriatal network thus influences synaptic plasticity and motor skill learning.

Huntingtin regulates calcium fluxes in skeletal muscle.

The expression of the Huntingtin protein, well known for its involvement in the neurodegenerative Huntington's disease, has been confirmed in skeletal muscle. The impact of HTT deficiency was studied in human skeletal muscle cell lines and in a mouse model with inducible and muscle-specific HTT deletion. Characterization of calcium fluxes in the knock-out cell lines demonstrated a reduction in excitation-contraction (EC) coupling, related to an alteration in the coupling between the dihydropyridine receptor and the ryanodine receptor, and an increase in the amount of calcium stored within the sarcoplasmic reticulum, linked to the hyperactivity of store-operated calcium entry (SOCE). Immunoprecipitation experiments demonstrated an association of HTT with junctophilin 1 (JPH1) and stromal interaction molecule 1 (STIM1), both providing clues on the functional effects of HTT deletion on calcium fluxes. Characterization of muscle strength and muscle anatomy of the muscle-specific HTT-KO mice demonstrated that HTT deletion induced moderate muscle weakness and mild muscle atrophy associated with histological abnormalities, similar to the phenotype observed in tubular aggregate myopathy. Altogether, this study points toward the hypotheses of the involvement of HTT in EC coupling via its interaction with JPH1, and on SOCE via its interaction with JPH1 and/or STIM1.

Neuronal architectures with axo-dendritic polarity above silicon nanowires.

An approach is developped to gain control over the polarity of neuronal networks at the cellular level by physically constraining cell development by the use of micropatterns. It is demonstrated that the position and path of individual axons, the cell extension that propagates the neuron output signal, can be chosen with a success rate higher than 85%. This allows the design of small living computational blocks above silicon nanowires.

Short-term intermittent hypoxia induces simultaneous systemic insulin resistance and higher cardiac contractility in lean mice.

BACKGROUND: Intermittent hypoxia (IH) is the major feature of obstructive sleep apnea syndrome, well-known to induce cardiometabolic complications. We previously demonstrated that IH induces hyperinsulinemia and associated altered insulin signaling in adipose tissue, liver, and skeletal muscle, but impact of IH on cardiac insulin signaling and functional/structural consequences remains unknown. Therefore, the aims of this study were to investigate in both lean and obese mice the effects of chronic IH on the following: (1) cardiac insulin signaling and (2) cardiac remodeling and function. METHODS: C57BL/6 J male mice were fed low-fat (LFD) or high-fat (HFD) diet for 20 weeks, and exposed to IH (21-5% FiO2, 60 s cycle, 8 h/day) or normoxia (N) for the last 6 weeks. Systemic insulin sensitivity was evaluated by an insulin tolerance test. Cardiac remodeling and contractile function were assessed by cardiac ultrasonography. Ultimately, hearts were withdrawn for biochemical and histological analysis. RESULTS: In LFD mice, IH-induced hyperinsulinemia and systemic insulin resistance that were associated with increased phosphorylations of cardiac insulin receptor and Akt on Tyr1150 and Ser473 residues, respectively. In addition, IH significantly increased cardiac interstitial fibrosis and cardiac contractility. In the HFD group, IH did not exert any additional effect, nor on insulin/Akt signaling, nor on cardiac remodeling and function. CONCLUSION: Our study suggests that, despite systemic insulin resistance, IH exposure mediates an adaptive cardiac response in lean but not in obese mice. Further studies are needed to investigate which specific mechanisms are involved and to determine the long-term evolution of cardiac responses to IH.

Triadin deletion induces impaired skeletal muscle function.

Triadin is a multiple proteins family, some isoforms being involved in muscle excitation-contraction coupling, and some having still unknown functions. To obtain clues on triadin functions, we engineered a triadin knock-out mouse line and characterized the physiological effect of triadin ablation on skeletal muscle function. These mice presented a reduced muscle strength, which seemed not to alter their survival and has been characterized in the present work. We first checked in these mice the expression level of the different proteins involved in calcium homeostasis and observed in fast muscles an increase in expression of dihydropyridine receptor, with a large reduction in calsequestrin expression. Electron microscopy analysis of KO muscles morphology demonstrated the presence of triads in abnormal orientation and a reduction in the sarcoplasmic reticulum terminal cisternae volume. Using calcium imaging on cultured myotubes, we observed a reduction in the total amount of calcium stored in the sarcoplasmic reticulum. Physiological studies have been performed to evaluate the influence of triadin deletion on skeletal muscle function. Muscle strength has been measured both on the whole animal model, using hang test or electrical stimulation combined with NMR analysis and strength measurement, or on isolated muscle using electrical stimulation. All the results obtained demonstrate an important reduction in muscle strength, indicating that triadin plays an essential role in skeletal muscle function and in skeletal muscle structure. These results indicate that triadin alteration leads to the development of a myopathy, which could be studied using this new animal model.

Dynamics of triadin, a muscle-specific triad protein, within sarcoplasmic reticulum subdomains.

In skeletal muscle, proteins of the calcium release complex responsible for the excitation-contraction (EC) coupling are exclusively localized in specific reticulum-plasma membrane (ER-PM) contact points named triads. The CRC protein triadin (T95) is localized in the sarcoplasmic reticulum (SR) subdomain of triads where it forms large multimers. However, the mechanisms leading to the steady-state accumulation of T95 in these specific areas of SR are largely unknown. To visualize T95 dynamics, fluorescent chimeras were expressed in triadin knockout myotubes, and their mobility was compared with the mobility of Sec61ß, a membrane protein of the SR unrelated to the EC coupling process. At all stages of skeletal muscle cells differentiation, we show a permanent flux of T95 diffusing in the SR membrane. Moreover, we find evidence that a longer residence time in the ER-PM contact point is due to the transmembrane domain of T95 resulting in an overall triad localization.

AutoNeuriteJ: An ImageJ plugin for measurement and classification of neuritic extensions.

Morphometry characterization is an important procedure in describing neuronal cultures and identifying phenotypic differences. This task usually requires labor-intensive measurements and the classification of numerous neurites from large numbers of neurons in culture. To automate these measurements, we wrote AutoNeuriteJ, an imageJ/Fiji plugin that measures and classifies neurites from a very large number of neurons. We showed that AutoNeuriteJ is able to detect variations of neuritic growth induced by several compounds known to affect the neuronal growth. In these experiments measurement of more than 5000 mouse neurons per conditions was obtained within a few hours. Moreover, by analyzing mouse neurons deficient for the microtubule associated protein 6 (MAP6) and wild type neurons we illustrate that AutoNeuriteJ is capable to detect subtle phenotypic difference in axonal length. Overall the use of AutoNeuriteJ will provide rapid, unbiased and accurate measurement of neuron morphologies.

Variations in the TRPV1 gene are associated to exertional heat stroke.

OBJECTIVES: Exertional Heat Stroke (EHS) is one of the top three causes of sudden death in athletes. Extrinsic and intrinsic risk factors have been identified but the genetic causes still remain unclear. Our aim was to identify genes responsible for EHS, which is a necessary step to identify patients at risk and prevent crises. DESIGN: Genetic and functional laboratory studies METHODS: Whole Exome Sequencing (WES) was performed to search for candidate genes in a cohort of 15 soldiers who had a documented EHS episode. In silico and in vitro functional studies were performed to evaluate the effect of mutations identified in the candidate gene TRPV1. RESULTS: WES led to the identification of two missense variations in the TRPV1 gene. These variations were very rare or unreported in control databases and located in critical domains of the protein. In vitro functional studies revealed that both variations induce a strong modification of the channel response to one of its natural agonist, the capsaicin. CONCLUSIONS: We evidenced mutations altering channel properties of the TRPV1 gene and demonstrated that TRPV1, which is involved in thermoregulation and nociception, is a new candidate gene for EHS. Our data provide the bases to explore genetic causes and molecular mechanisms governing the pathophysiology of EHS.

Ferroquine, an ingenious antimalarial drug: thoughts on the mechanism of action.

Ferroquine (FQ or SR97193) is a novel antimalarial drug candidate, currently in development at Sanofi-Aventis. In contrast to conventional drugs, FQ is the first organometallic drug: a ferrocenyl group covalently flanked by a 4-aminoquinoline and a basic alkylamine. FQ is able to overcome the CQ resistance problem, an important limit to the control of Plasmodium falciparum, the principal causative agent of malaria. After fifteen years of effort, it is now possible to propose a multifactorial mechanism of action of FQ by its capacity to target lipids, to inhibit the formation of hemozoin and to generate reactive oxygen species.

Deletion of the microtubule-associated protein 6 (MAP6) results in skeletal muscle dysfunction.

BACKGROUND: The skeletal muscle fiber has a specific and precise intracellular organization which is at the basis of an efficient muscle contraction. Microtubules are long known to play a major role in the function and organization of many cells, but in skeletal muscle, the contribution of the microtubule cytoskeleton to the efficiency of contraction has only recently been studied. The microtubule network is dynamic and is regulated by many microtubule-associated proteins (MAPs). In the present study, the role of the MAP6 protein in skeletal muscle organization and function has been studied using the MAP6 knockout mouse line. METHODS: The presence of MAP6 transcripts and proteins was shown in mouse muscle homogenates and primary culture using RT-PCR and western blot. The in vivo evaluation of muscle force of MAP6 knockout (KO) mice was performed on anesthetized animals using electrostimulation coupled to mechanical measurement and multimodal magnetic resonance. The impact of MAP6 deletion on microtubule organization and intracellular structures was studied using immunofluorescent labeling and electron microscopy, and on calcium release for muscle contraction using Fluo-4 calcium imaging on cultured myotubes. Statistical analysis was performed using Student's t test or the Mann-Whitney test. RESULTS: We demonstrate the presence of MAP6 transcripts and proteins in skeletal muscle. Deletion of MAP6 results in a large number of muscle modifications: muscle weakness associated with slight muscle atrophy, alterations of microtubule network and sarcoplasmic reticulum organization, and reduction in calcium release. CONCLUSION: Altogether, our results demonstrate that MAP6 is involved in skeletal muscle function. Its deletion results in alterations in skeletal muscle contraction which contribute to the global deleterious phenotype of the MAP6 KO mice. As MAP6 KO mouse line is a model for schizophrenia, our work points to a possible muscle weakness associated to some forms of schizophrenia.

A key function for microtubule-associated-protein 6 in activity-dependent stabilisation of actin filaments in dendritic spines.

Emerging evidence indicates that microtubule-associated proteins (MAPs) are implicated in synaptic function; in particular, mice deficient for MAP6 exhibit striking deficits in plasticity and cognition. How MAP6 connects to plasticity mechanisms is unclear. Here, we address the possible role of this protein in dendritic spines. We find that in MAP6-deficient cortical and hippocampal neurons, maintenance of mature spines is impaired, and can be restored by expressing a stretch of the MAP6 sequence called Mc modules. Mc modules directly bind actin filaments and mediate activity-dependent stabilisation of F-actin in dendritic spines, a key event of synaptic plasticity. In vitro, Mc modules enhance actin filament nucleation and promote the formation of stable, highly ordered filament bundles. Activity-induced phosphorylation of MAP6 likely controls its transfer to the spine cytoskeleton. These results provide a molecular explanation for the role of MAP6 in cognition, enlightening the connection between cytoskeletal dysfunction, synaptic impairment and neuropsychiatric illnesses.

Short- and long-term efficacy of electroconvulsive stimulation in animal models of depression: The essential role of neuronal survival.

BACKGROUND: Severe and medication-resistant psychiatric diseases, such as major depressive disorder, bipolar disorder or schizophrenia, can be effectively and rapidly treated by electroconvulsive therapy (ECT). Despite extensive long-standing clinical use, the neurobiological mechanisms underlying the curative action of ECT remain incompletely understood. OBJECTIVE: Unravel biological basis of electroconvulsive stimulation (ECS) efficacy, the animal equivalent of ECT. METHODS: Using MAP6 KO mouse, a genetic model that constitutively exhibits features relevant to some aspects of depression; we analyzed the behavioral and biological consequences of ECS treatment alone (10 stimulations over a 2-week period) and associated with a continuation protocol (2 stimulations per week for 5 weeks). RESULTS: ECS treatment had a beneficial effect on constitutive behavioral defects. We showed that behavioral improvement is associated with a strong increase in the survival and integration of neurons born before ECS treatment. Retroviral infection revealed the larger number of integrated neurons to exhibit increased dendritic complexity and spine density, as well as remodeled synapses. Furthermore, our results show that ECS triggers a cortical increase in synaptogenesis. A sustained newborn neuron survival rate, induced by ECS treatment, is associated with the behavioral improvement, but relapse occurred 40 days after completing the ECS treatment. However, a 5-week continuation protocol following the initial ECS treatment led to persistent improvement of behavior correlated with sustained rate survival of newborn neurons. CONCLUSION: Altogether, these results reveal that increased synaptic connectivity and extended neuronal survival are key to the short and long-term efficacy of ECS.

3D imaging of the brain morphology and connectivity defects in a model of psychiatric disorders: MAP6-KO mice.

In the central nervous system, microtubule-associated protein 6 (MAP6) is expressed at high levels and is crucial for cognitive abilities. The large spectrum of social and cognitive impairments observed in MAP6-KO mice are reminiscent of the symptoms observed in psychiatric diseases, such as schizophrenia, and respond positively to long-term treatment with antipsychotics. MAP6-KO mice have therefore been proposed to be a useful animal model for these diseases. Here, we explored the brain anatomy in MAP6-KO mice using high spatial resolution 3D MRI, including a volumetric T1w method to image brain structures, and Diffusion Tensor Imaging (DTI) for white matter fiber tractography. 3D DTI imaging of neuronal tracts was validated by comparing results to optical images of cleared brains. Changes to brain architecture included reduced volume of the cerebellum and the thalamus and altered size, integrity and spatial orientation of some neuronal tracks such as the anterior commissure, the mammillary tract, the corpus callosum, the corticospinal tract, the fasciculus retroflexus and the fornix. Our results provide information on the neuroanatomical defects behind the neurological phenotype displayed in the MAP6-KO mice model and especially highlight a severe damage of the corticospinal tract with defasciculation at the location of the pontine nuclei.