The AMPK-related kinase NUAK1 controls cortical axons branching by locally modulating mitochondrial metabolic functions.

The cellular mechanisms underlying axonal morphogenesis are essential to the formation of functional neuronal networks. We previously identified the autism-linked kinase NUAK1 as a central regulator of axon branching through the control of mitochondria trafficking. However, (1) the relationship between mitochondrial position, function and axon branching and (2) the downstream effectors whereby NUAK1 regulates axon branching remain unknown. Here, we report that mitochondria recruitment to synaptic boutons supports collateral branches stabilization rather than formation in mouse cortical neurons. NUAK1 deficiency significantly impairs mitochondrial metabolism and axonal ATP concentration, and upregulation of mitochondrial function is sufficient to rescue axonal branching in NUAK1 null neurons in vitro and in vivo. Finally, we found that NUAK1 regulates axon branching through the mitochondria-targeted microprotein BRAWNIN. Our results demonstrate that NUAK1 exerts a dual function during axon branching through its ability to control mitochondrial distribution and metabolic activity.

Deciphering the Role of Klf10 in the Cerebellum.

Recent studies have demonstrated a new role for Klf10, a Krüppel-like transcription factor, in skeletal muscle, specifically relating to mitochondrial function. Thus, it was of interest to analyze additional tissues that are highly reliant on optimal mitochondrial function such as the cerebellum and to decipher the role of Klf10 in the functional and structural properties of this brain region. In vivo (magnetic resonance imaging and localized spectroscopy, behavior analysis) and in vitro (histology, spectroscopy analysis, enzymatic activity) techniques were applied to comprehensively assess the cerebellum of wild type (WT) and Klf10 knockout (KO) mice. Histology analysis and assessment of locomotion revealed no significant difference in Klf10 KO mice. Diffusion and texture results obtained using MRI revealed structural changes in KO mice characterized as defects in the organization of axons. These modifications may be explained by differences in the levels of specific metabolites (myo-inositol, lactate) within the KO cerebellum. Loss of Klf10 expression also led to changes in mitochondrial activity as reflected by a significant increase in the activity of citrate synthase, complexes I and IV. In summary, this study has provided evidence that Klf10 plays an important role in energy production and mitochondrial function in the cerebellum.

In Utero Cortical Electroporation of Plasmids in the Mouse Embryo.

The cerebral cortex is composed of an exquisitely complex network of interconnected neurons supporting the higher cognitive functions of the brain. Here, we provide a fully detailed, step-by-step protocol to perform in utero cortical electroporation of plasmids, a simple surgical procedure designed to manipulate gene expression in a subset of glutamatergic pyramidal cortical neurons in vivo. This method has been used to visualize defects in neuronal migration, axon projections, terminal axon branching, or dendrite and synapse development. For complete details on the use and execution of this protocol, please refer to Courchet et al., 2013, Mairet-Coello et al., 2013 or Shimojo et al. (2015).

3Rs-based optimization of mice behavioral testing: The habituation/dishabituation olfactory test.

BACKGROUND: There is clear evidence that most of the paradigms that are used in the field of behavioral neuroscience suffer from a lack of reliability mainly because of oversimplification of both testing procedures and interpretations. In the present study we show how an already existing behavioral test, the olfactory habituation / dishabituation task, can be optimized in such a way that animal number and animal distress could be minimized, number/confidence of behavioral outcomes and number of explored behavioral dimensions could be increased. NEW METHOD: We used ethologically relevant technical and procedural changes associated with videotracking-based automated quantification of sniffing behavior to validate our new setup. Mainly internal and construct validity were challenged through the implementation of a series of simple experiments. RESULTS: We show that the new version of the test: 1) has very good within and inter laboratory replicability, 2) is sensitive to some environmental / experimental factors while insensitive to others, 3) allows investigating hedonism, both state and trait anxiety, efficacy of anxiolytic molecules, acute stress, mental retardation-related social impairments and learning and memory. 4) We also show that interest for both nonsocial and social odors is stable over time which makes repetitive testing possible. CONCLUSIONS: This work paves the way for future studies showing how behavioral tests / procedures may be improved by using ethologically relevant changes, in order to question laboratory animals more adequately. Refining behavioral tests may considerably increase predictivity of preclinical tests and, ultimately, help reinforcing translational research.

Visual Behavior Impairments as an Aberrant Sensory Processing in the Mouse Model of Fragile X Syndrome.

Fragile X Syndrome (FXS), the most common inherited form of human intellectual disability (ID) associated with autistic-like behaviors, is characterized by dys-sensitivity to sensory stimuli, especially vision. In the absence of Fragile Mental Retardation Protein (FMRP), both retinal and cerebral structures of the visual pathway are impaired, suggesting that perception and integration of visual stimuli are altered. However, behavioral consequences of these defects remain unknown. In this study, we used male Fmr1 -/y mice to further define visual disturbances from a behavioral perspective by focusing on three traits characterizing visual modality: perception of depth, contrasts and movements. We performed specific tests (Optomotor Drum, Visual Cliff) to evaluate these visual modalities, their evolution from youth to adulthood, and to assess their involvement in a cognitive task. We show that Fmr1 -/y mice exhibit alteration in their visual skills, displaying impaired perspective perception, a drop in their ability to understand a moving contrasted pattern, and a defect in contrasts discrimination. Interestingly, Fmr1 -/y phenotypes remain stable over time from adolescence to late adulthood. Besides, we report that color and shape are meaningful for the achievement of a cognitive test involving object recognition. Altogether, these results underline the significance of visual behavior alterations in FXS conditions and relevance of assessing visual skills in neuropsychiatric models before performing behavioral tasks, such as cognitive assessments, that involve visual discrimination.

Haploinsufficiency of autism spectrum disorder candidate gene NUAK1 impairs cortical development and behavior in mice.

Recently, numerous rare de novo mutations have been identified in patients diagnosed with autism spectrum disorders (ASD). However, despite the predicted loss-of-function nature of some of these de novo mutations, the affected individuals are heterozygous carriers, which would suggest that most of these candidate genes are haploinsufficient and/or lead to expression of dominant-negative forms of the protein. Here, we tested this hypothesis with the candidate ASD gene Nuak1 that we previously identified for its role in the development of cortical connectivity. We report that Nuak1 is haploinsufficient in mice with regard to its function in cortical development. Furthermore Nuak1+/- mice show a combination of abnormal behavioral traits ranging from defective spatial memory consolidation, defects in social novelty (but not social preference) and abnormal sensorimotor gating. Overall, our results demonstrate that Nuak1 haploinsufficiency leads to defects in the development of cortical connectivity and a complex array of behavorial deficits.

Multiple effects of the herbicide glufosinate-ammonium and its main metabolite on neural stem cells from the subventricular zone of newborn mice.

The globally used herbicide glufosinate-ammonium (GLA) is structurally analogous to the excitatory neurotransmitter glutamate, and is known to interfere with cellular mechanisms involved in the glutamatergic system. In this report, we used an in vitro model of murine primary neural stem cell culture to investigate the neurotoxicity of GLA and its main metabolite, 4-methylphosphinico-2-oxobutanoic acid (PPO). We demonstrated that GLA and PPO disturb ependymal wall integrity in the ventricular-subventricular zone (V-SVZ) and alter the neuro-glial differentiation of neural stem cells. GLA and PPO impaired the formation of cilia, with reduced Celsr2 expression after PPO exposure. GLA promoted the differentiation of neuronal and oligodendroglial cells while PPO increased B1 cell population and impaired neuronal fate of neural stem cells. These results confirm our previous in vivo report that developmental exposure to GLA alters neurogenesis in the SVZ, and neuroblast migration along the rostral migratory stream. They also highlight the importance of investigating the toxicity of pesticide degradation products. Indeed, not only GLA, but also its metabolite PPO disrupts V-SVZ homeostasis and provides a novel cellular mechanism underlying GLA-induced neurodevelopmental toxicity. Furthermore, we were able to demonstrate a neurotoxic activity of a metabolite of GLA different from that of GLA active substance for the very first time.

Early Retinal Defects in Fmr1-/y Mice: Toward a Critical Role of Visual Dys-Sensitivity in the Fragile X Syndrome Phenotype?

Fragile X Syndrome (FXS) is caused by a deficiency in Fragile X Mental Retardation Protein (FMRP) leading to global sensorial abnormalities, among which visual defects represent a critical part. These visual defects are associated with cerebral neuron immaturity especially in the primary visual cortex. However, we recently demonstrated that retinas of adult Fmr1-/y mice, the FXS murine model, present molecular, cellular and functional alterations. However, no data are currently available on the evolution pattern of such defects. As retinal stimulation through Eye Opening (EO) is a crucial signal for the cerebral visual system maturation, we questioned the precocity of molecular and functional retinal phenotype. To answer this question, we studied the retinal molecular phenotype of Fmr1-/y mice before EO until adult age and the consequences of the retinal loss of Fmrp on retinal function in young and adult mice. We showed that retinal molecular defects are present before EO and remain stable at adult age, leading to electrophysiological impairments without any underlying structural changes. We underlined that loss of Fmrp leads to a wide range of defects in the retina, settled even before EO. Our work demonstrates a critical role of the sensorial dysfunction in the Fmr1-/y mice overall phenotype, and provides evidence that altered peripheral perception is a component of the sensory processing defect in FXS conditions.

Perinatal Exposure to the Cyanotoxin ß-N-Méthylamino-L-Alanine (BMAA) Results in Long-Lasting Behavioral Changes in Offspring-Potential Involvement of DNA Damage and Oxidative Stress.

We recently demonstrated that perinatal exposure to the glutamate-related herbicide, glufosinate ammonium, has deleterious effects on neural stem cell (NSC) homeostasis within the sub-ventricular zone (SVZ), probably leading to ASD-like symptoms in offspring later in life. In the present study, we aimed to investigate whether perinatal exposure to another glutamate-related toxicant, the cyanobacterial amino acid ß-N-methylamino-L-alanine (BMAA), might also trigger neurodevelopmental disturbances. With this aim, female mice were intranasally exposed to low doses of BMAA, 50 mg kg-1 three times a week from embryonic days 7-10 to postnatal day 21. Behavioral analyses were performed during the offspring's early life and during adulthood. Developmental analyses revealed that perinatal exposure to BMAA hastened the appearance of some reflexes and communicative skills. BMAA-exposed offspring displayed sex-dependent changes in emotional cognition shortly after exposure. Later in life, the female offspring continued to express emotional defects and to display abnormal sociability, while males were less affected. To assess whether early exposure to BMAA had deleterious effects on NSC homeostasis, we exposed mice NSCs to 1 and 3 mM BMAA during 24 h. We found that BMAA-exposed NSCs produced high levels of ROS, highlighting the ability of BMAA to induce oxidative stress. We also showed that BMAA exposure increased the number of γH2AX/53BP1 foci per nucleus, suggesting that BMAA-induced DNA damage in NSCs. Collectively, this data strongly suggests that perinatal exposure to the cyanobacteria BMAA, even at low doses, results in neurobehavioral disturbances during both the postnatal period and adulthood. This is considered to be underpinned at the cellular level through dysregulation of NSC homeostasis in the developing brain.