Etude fonctionnelle du système HGF/Met au cours du développement normal et pathologique du système nerveux central

Les signaux participant au développement embryonnaire du système nerveux central peuvent à nouveau intervenir à l âge adulte, notamment lors d une pathologie. Nous avons analysé si le couple HGF/Met peut avoir de telles fonctions. Aussi, nous avons généré des souris porteuses d une mutation conditionnelle de Met spécifique du système nerveux. Nous avons montré que 1) dans le cerveau, Met participe à l établissement de l équilibre entre circuits excitateurs et inhibiteurs et 2) dans la moelle épinière, HGF/Met sont requis in vivo pour la survie d un sous-type de motoneurones. Nous avons aussi montré l intérêt du rôle neuroprotecteur de Met dans deux conditions pathologiques : un modèle murin de la sclérose latérale amyotrophique et dans la rétine après axotomie du nerf optique. Cette étude a montré que le système HGF/Met possède des propriétés trophiques spécifiques lors du développement des MNs et permet dans le cas d une neurodégénérescence de protéger les neurones contre l apoptose.Signals involved in development of the embryonic central nervous system can also play a role during adulthood, particularly in some pathologies. In order to analyze whether the HGF/Met system would have such functions, we generated met conditional mutant mice in which met is specifically removed in the nervous system. Using these mice, we have shown that 1) in the brain, Met is part of a system which controls the balance between excitatory and inhibitory circuits and 2) in the spinal cord, HGF/Met signaling is required in vivo for the survival of a specific subtype of motor neurons (MNs). We have also demonstrated the neuroprotective effect of HGF/Met in two pathological conditions such as the amyotrophic lateral sclerosis and after optic nerve axotomy. In conclusion, this work has allowed us to show the trophic properties of HGF/Met during normal development of specific subtypes of MNs, and its neuroprotective effect in neurodegenerative mouse modelsAIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Autism-relevant social abnormalities and cognitive deficits in engrailed-2 knockout mice.

ENGRAILED 2 (En2), a homeobox transcription factor, functions as a patterning gene in the early development and connectivity of rodent hindbrain and cerebellum, and regulates neurogenesis and development of monoaminergic pathways. To further understand the neurobiological functions of En2, we conducted neuroanatomical expression profiling of En2 wildtype mice. RTQPCR assays demonstrated that En2 is expressed in adult brain structures including the somatosensory cortex, hippocampus, striatum, thalamus, hypothalamus and brainstem. Human genetic studies indicate that EN2 is associated with autism. To determine the consequences of En2 mutations on mouse behaviors, including outcomes potentially relevant to autism, we conducted comprehensive phenotyping of social, communication, repetitive, and cognitive behaviors. En2 null mutants exhibited robust deficits in reciprocal social interactions as juveniles and adults, and absence of sociability in adults, replicated in two independent cohorts. Fear conditioning and water maze learning were impaired in En2 null mutants. High immobility in the forced swim test, reduced prepulse inhibition, mild motor coordination impairments and reduced grip strength were detected in En2 null mutants. No genotype differences were found on measures of ultrasonic vocalizations in social contexts, and no stereotyped or repetitive behaviors were observed. Developmental milestones, general health, olfactory abilities, exploratory locomotor activity, anxiety-like behaviors and pain responses did not differ across genotypes, indicating that the behavioral abnormalities detected in En2 null mutants were not attributable to physical or procedural confounds. Our findings provide new insight into the role of En2 in complex behaviors and suggest that disturbances in En2 signaling may contribute to neuropsychiatric disorders marked by social and cognitive deficits, including autism spectrum disorders

Autism-relevant social abnormalities and cognitive deficits in engrailed-2 knockout mice.

ENGRAILED 2 (En2), a homeobox transcription factor, functions as a patterning gene in the early development and connectivity of rodent hindbrain and cerebellum, and regulates neurogenesis and development of monoaminergic pathways. To further understand the neurobiological functions of En2, we conducted neuroanatomical expression profiling of En2 wildtype mice. RTQPCR assays demonstrated that En2 is expressed in adult brain structures including the somatosensory cortex, hippocampus, striatum, thalamus, hypothalamus and brainstem. Human genetic studies indicate that EN2 is associated with autism. To determine the consequences of En2 mutations on mouse behaviors, including outcomes potentially relevant to autism, we conducted comprehensive phenotyping of social, communication, repetitive, and cognitive behaviors. En2 null mutants exhibited robust deficits in reciprocal social interactions as juveniles and adults, and absence of sociability in adults, replicated in two independent cohorts. Fear conditioning and water maze learning were impaired in En2 null mutants. High immobility in the forced swim test, reduced prepulse inhibition, mild motor coordination impairments and reduced grip strength were detected in En2 null mutants. No genotype differences were found on measures of ultrasonic vocalizations in social contexts, and no stereotyped or repetitive behaviors were observed. Developmental milestones, general health, olfactory abilities, exploratory locomotor activity, anxiety-like behaviors and pain responses did not differ across genotypes, indicating that the behavioral abnormalities detected in En2 null mutants were not attributable to physical or procedural confounds. Our findings provide new insight into the role of En2 in complex behaviors and suggest that disturbances in En2 signaling may contribute to neuropsychiatric disorders marked by social and cognitive deficits, including autism spectrum disorders

Engrailed-2 (En2) deletion produces multiple neurodevelopmental defects in monoamine systems, forebrain structures and neurogenesis and behavior

Many genes involved in brain development have been associated with human neurodevelopmental disorders, but underlying pathophysiological mechanisms remain undefined. Human genetic and mouse behavioral analyses suggest that ENGRAILED-2 (EN2) contributes to neurodevelopmental disorders, especially autism spectrum disorder. In mouse, En2 exhibits dynamic spatiotemporal expression in embryonic mid-hindbrain regions where monoamine neurons emerge. Considering their importance in neuropsychiatric disorders, we characterized monoamine systems in relation to forebrain neurogenesis in En2-knockout (En2-KO) mice. Transmitter levels of serotonin, dopamine and norepinephrine (NE) were dysregulated from Postnatal day 7 (P7) to P21 in En2-KO, though NE exhibited the greatest abnormalities. While NE levels were reduced ∼35% in forebrain, they were increased 40 -: 75% in hindbrain and cerebellum, and these patterns paralleled changes in locus coeruleus (LC) fiber innervation, respectively. Although En2 promoter was active in Embryonic day 14.5 -: 15.5 LC neurons, expression diminished thereafter and gene deletion did not alter brainstem NE neuron numbers. Significantly, in parallel with reduced NE levels, En2-KO forebrain regions exhibited reduced growth, particularly hippocampus, where P21 dentate gyrus granule neurons were decreased 16%, suggesting abnormal neurogenesis. Indeed, hippocampal neurogenic regions showed increased cell death (+77%) and unexpectedly, increased proliferation. Excess proliferation was restricted to early Sox2/Tbr2 progenitors whereas increased apoptosis occurred in differentiating (Dcx) neuroblasts, accompanied by reduced newborn neuron survival. Abnormal neurogenesis may reflect NE deficits because intra-hippocampal injections of β-adrenergic agonists reversed cell death. These studies suggest that disruption of hindbrain patterning genes can alter monoamine system development and thereby produce forebrain defects that are relevant to human neurodevelopmental disorders

Adult <i>En2</i>−/− exhibit absence of sociability and deficits in male-female social interactions.

<p>Social approach was tested in two separate cohorts using our automated three-chambered apparatus. Cohort 1: (<b>A</b>) <i>En2</i>+/+ and +/− displayed sociability, defined as spending more time in the chamber with the novel mouse than in the chamber with the novel object. <i>En2</i>−/− did not spend more time in the novel mouse chamber as compared to the novel object chamber, meeting the definition of lack of sociability for this task. (<b>B</b>) <i>En2</i>+/+ and +/− spent more time sniffing the novel mouse than the novel object. <i>En2</i>−/− did not spend more time sniffing the novel mouse than the novel object, meeting the definition of lack of sociability on this more sensitive parameter of social interaction, and confirming results from the chamber time parameter investigation. (<b>C</b>) No genotype differences were found for time spent in each chamber during the habituation phase. Cohort 2: (<b>D</b>) Similar lack of sociability was seen in <i>En2</i>−/− mice for time spent in the novel mouse chamber vs. the novel object chamber. (<b>E</b>) <i>En2</i>−/− mice again failed to spend more time sniffing the novel mouse vs. the novel object. (<b>F</b>) Time spent in each chamber during the habituation phase was not different between genotypes. Cohort 1: N = 16+/+, N = 16+/−, N = 15−/−; Cohort 2: N = 10+/+, N = 13+/−, N = 14−/−. *p<05 vs. novel object. Reciprocal social interactions and ultrasonic vocalizations (USVs) were measured in male <i>En2</i> mice during interaction with an unfamiliar estrus female mouse. (<b>G</b>) <i>En2</i>−/− males spent less time engaged in sniffing the body and anogenital regions of the female as compared to +/+ males. (<b>H</b>) The total number of USVs emitted during the test session did not differ between genotypes. (<b>I</b>) No genotype differences were found for bouts of test cage exploration during the 5-minute test session. N = 10+/+, N = 13+/−, N = 13−/−. *p<05 vs. +/+.</p

Statistical results for olfactory habituation/dishabituation.

<p>Summary of statistical results of the olfactory habituation/dishabituation test. Data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040914#pone-0040914-g010" target="_blank">Figure 10</a>.</p

Juvenile <i>En2</i> mutant mice display fewer reciprocal social interactions, as replicated in two cohorts.

<p>Cohort 1: As compared to wildtype littermates (+/+), <i>En2</i> null mutant mice (−/−) exhibited fewer bouts of (<b>A</b>) nose-to-nose sniffing. <i>En2</i>+/− and −/− mice displayed fewer bouts of (<b>B</b>) anogenital sniffing, and (<b>C</b>) following as compared to +/+ controls. No significant genotype differences were detected in (<b>D</b>) front approach, (<b>E</b>) self-grooming, and (<b>F</b>) exploration. Cohort 2: As compared to +/+, +/− and −/− exhibited fewer bouts of (<b>G</b>) nose-to-nose sniffing and (<b>H</b>) anogenital sniffing. <i>En2</i>−/− mice exhibited fewer bouts of (<b>J</b>) front approach as compared to +/+. No significant genotype differences were detected for (<b>I</b>) following behaviors, (<b>K</b>) self-grooming, or (<b>L</b>) arena exploration. Cohort 1: N = 15+/+; N = 15+/−; N = 16−/−; Cohort 2: N = 14+/+; N = 15+/−; N = 10−/−. *p<05 vs. +/+.</p

Cognitive deficits in <i>En2</i> null mutants.

<p>Cumulative time spent freezing during the fear conditioning test sessions, as quantified by the VideoFreeze software, was converted to percent time freezing for data analysis and presentation. (<b>A</b>) Despite normal postshock freezing during training, <i>En2</i>−/− exhibited significantly less freezing than <i>En2</i>+/+ and +/− mice upon testing of contextual and cued fear memory. N = 23+/+, N = 23+/−, N = 20−/−. *p<.005 vs. +/+ and +/−. (<b>B</b>) In the novel object recognition test, a lack of innate object preference was observed for <i>En2</i>+/+, +/− and −/− mice during the familiarization phase of the task. (<b>C</b>) <i>En2</i>+/+ displayed novel object recognition memory, defined as spending more time sniffing the novel object as compared to the familiar object. <i>En2</i>+/− exhibited a trend towards significant preference for the novel object, whereas −/− failed to display a preference for the novel object. N = 16+/+, N = 17+/−, N = 16−/−. *p<0.05 vs. familiar object. (<b>D</b>) In the Morris water maze, <i>En2</i>−/− showed longer latencies to reach the hidden platform during training trials as compared to +/+. *p<u><</u>.01 vs. +/+. (<b>E</b>) In the probe trial, +/+ and +/− mice showed selective quadrant search with a greater percentage of time spent in the training quadrant as compared to the non-trained quadrants, while −/− failed to show selective search. (<b>F</b>) <i>En2</i>+/+ displayed a greater proportion of platform crossings in the trained quadrant as compared to the analogous locations in the non-trained quadrants, whereas +/− and −/− did not. N = 16+/+, N = 13+/−, N = 13−/−. *p<05 vs. non-trained quadrant.</p

RTQPCR analysis demonstrates that <i>En2</i> is expressed in multiple adult brain structures.

<p>Average ▵Ct values with standard error are shown for the following brain structures and tissue: somatosensory cortex (ssctx), hippocampus (hippo), striatum, hypothalamus (hypoth), thalamus (thal), colliculi (coll), cerebellum (cereb), brainstem (bstem), amygdala (amyg), visual cortex (vctx), prefrontal cortex (pctx), olfactory bulb (ob), and hindlimb muscle (ms). Lower ▵Ct values indicate high gene expression, whereas higher values reflect lower levels. nd  =  none detected.</p

Statistical results for selected parameters of open field locomotor activity.

<p>Summary of statistical results of selected parameters of open field locomotor activity. Data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040914#pone-0040914-g009" target="_blank">Figure 9</a>.</p