Extracellular signal-regulated kinase 2 has duality in function between neuronal and astrocyte expression following neonatal hypoxic-ischaemic cerebral injury

Hypoxia–ischaemia (HI) is a major cause of neonatal brain injury resulting in cerebral palsy, epilepsy, cognitive impairment and other neurological disabilities. The role of extracellular signal‐regulated kinase (ERK) isoforms and their mitogen‐activated protein kinase kinase (MEK)‐dependent phosphorylation in HI has previously been explored but remains unresolved at cellular level. This is pertinent given the growing awareness of the role of non‐neuronal cells in neuroprotection. Using a modified Rice–Vannucci model of HI in the neonatal mouse we observed time‐ and cell‐dependent ERK phosphorylation (pERK), with strongly up‐regulated pERK immunoreactivity first in periventricular white matter axons within 15–45 min of HI, followed by forebrain astrocytes and neurons (1–4 h post‐HI), and return to baseline by 16 h. We explored the effects of pharmacological ERK blockade through the MEK inhibitor SL327 on neonatal HI‐brain damage following HI alone (30 or 60 min) or lipopolysaccharide (LPS)‐sensitised HI insult (30 min). Global inhibition of ERK phosphorylation with systemically applied SL327 abolished forebrain pERK immunoreactivity, and significantly reduced cell death and associated microglial activation at 48 h post‐HI. We then explored the effects of cell‐specific ERK2 deletion alone or in combination with global ERK1 knockout under the same conditions of HI insult. Neuronal ERK2 deletion strongly decreased infarct size, neuronal cell death and microglial activation in grey matter following both HI alone or LPS‐sensitised HI. ERK1 deletion attenuated the protective effect of neuronal ERK2 deletion. Removal of astroglial ERK2 produced a reverse response, with a 3‐ to 4‐fold increase in microglial activation and cell death. Our data suggest a cell‐specific and time‐dependent role of ERK in neonatal HI, with a predominant, neurotoxic effect of neuronal ERK2, which is counteracted by neuroprotection by ERK1 and astrocytic ERK2. Overall, global pharmacological inhibition of ERK phosphorylation is strongly neuroprotective

Neuronal c-Jun is required for successful axonal regeneration, but the effects of phosphorylation of its N-terminus are moderate.

Although neural c-Jun is essential for successful peripheral nerve regeneration, the cellular basis of this effect and the impact of c-Jun activation are incompletely understood. In the current study, we explored the effects of neuron-selective c-Jun deletion, substitution of serine 63 and 73 phosphoacceptor sites with non-phosphorylatable alanine, and deletion of Jun N-terminal kinases 1, 2 and 3 in mouse facial nerve regeneration. Removal of the floxed c-jun gene in facial motoneurons using cre recombinase under control of a neuron-specific synapsin promoter (junΔS) abolished basal and injury-induced neuronal c-Jun immunoreactivity, as well as most of the molecular responses following facial axotomy. Absence of neuronal Jun reduced the speed of axonal regeneration following crush, and prevented most cut axons from reconnecting to their target, significantly reducing functional recovery. Despite blocking cell death, this was associated with a large number of shrunken neurons. Finally, junΔS mutants also had diminished astrocyte and microglial activation and T-cell influx, suggesting that these non-neuronal responses depend on the release of Jun-dependent signals from neighboring injured motoneurons. The effects of substituting serine 63 and 73 phosphoacceptor sites (junAA), or of global deletion of individual kinases responsible for N-terminal c-Jun phosphorylation were mild. junAA mutants showed decrease in neuronal cell size, a moderate reduction in post-axotomy CD44 levels and slightly increased astrogliosis. Deletion of Jun N-terminal kinase (JNK)1 or JNK3 showed delayed functional recovery; deletion of JNK3 also interfered with T-cell influx, and reduced CD44 levels. Deletion of JNK2 had no effect. Thus, neuronal c-Jun is needed in regeneration, but JNK phosphorylation of the N-terminus mostly appears to not be required for its function

Régulation du phénotype neuronal sérotoninergique in vitro par le brain-derived neurotrophic factor (BDNF) et l'AMPc

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Differential subcellular localization of the 5-HT 3 -A s receptor subunit in the rat central nervous system

International audienceFollowing the cloning and sequencing of the A subunit of the 5-HT3 receptor, two alternatively spliced isoforms, 5-HT3-AS and 5-HT3-AL, have been identified. In order to analyse the distribution of the receptor, a polyclonal antibody has been produced against the short form which is the most abundant in the central nervous system [Doucet et al. (2000) Neuroscience 95, 881-892]. As expected from the recognition of functional 5-HT3 receptors, immunostaining by this anti-5-HT3-R-AS antibody matched the distribution of the high-affinity 5-HT3 binding sites in the rat brain and spinal cord. 5-HT3-AS-like immunoreactivity was detected at low levels in the limbic system, particularly in the amygdala and the hippocampus, and in the frontal, piriform and entorhinal cortices. High levels of immunoreactivity were found in the brainstem, mainly in the nucleus tractus solitarius and the nucleus of the spinal tract of the trigeminal nerve, and in the dorsal horn of the spinal cord. At the ultrastructural level, immunostaining was generally found associated with axons and nerve terminals (70-80%) except in the hippocampus, where labelled dendrites were more abundant (56%). This preferential localization on nerve endings is consistent with the well-documented physiological role of 5-HT3 receptors in the control of neurotransmitter release. However, the different distribution in the hippocampus raises the question of whether differential addressing mechanisms exist for preferentially targeting 5-HT3 receptors to postsynaptic dendritic sites as compared to presynaptic nerve endings, depending on the nature of the neurons bearing these receptors

The extent of intrauterine growth restriction determines the severity of cerebral injury and neurobehavioural deficits in rodents

<div><p>Background</p><p>Cerebral Palsy (CP) is the most common physical pediatric neurodevelopmental disorder and spastic diplegic injury is its most frequent subtype. CP results in substantial neuromotor and cognitive impairments that have significant socioeconomic impact. Despite this, its underlying pathophysiological mechanisms and etiology remain incompletely understood. Furthermore, there is a need for clinically relevant injury models, which a) reflect the heterogeneity of the condition and b) can be used to evaluate new translational therapies. To address these key knowledge gaps, we characterized a chronic placental insufficiency (PI) model, using bilateral uterine artery ligation (BUAL) of dams. This injury model results in intrauterine growth restriction (IUGR) in pups, and animals recapitulate the human phenotype both in terms of neurobehavioural and anatomical deficits.</p><p>Methods</p><p>Effects of BUAL were studied using luxol fast blue (LFB)/hematoxylin & eosin (H&E) staining, immunohistochemistry, quantitative Magnetic Resonance Imaging (MRI), and Catwalk neurobehavioural tests.</p><p>Results</p><p>Neuroanatomical analysis revealed regional ventricular enlargement and corpus callosum thinning in IUGR animals, which was correlated with the extent of growth restriction. Olig2 staining revealed reductions in oligodendrocyte density in white and grey matter structures, including the corpus callosum, optic chiasm, and nucleus accumbens. The caudate nucleus, along with other brain structures such as the optic chiasm, internal capsule, septofimbrial and lateral septal nuclei, exhibited reduced size in animals with IUGR. The size of the pretectal nucleus was reduced only in moderately injured animals. MAG/NF200 staining demonstrated reduced myelination and axonal counts in the corpus callosum of IUGR animals. NeuN staining revealed changes in neuronal density in the hippocampus and in the thickness of hippocampal CA2 and CA3 regions. Diffusion weighted imaging (DWI) revealed regional white and grey matter changes at 3 weeks of age. Furthermore, neurobehavioural testing demonstrated neuromotor impairments in animals with IUGR in paw intensities, swing speed, relative print positions, and phase dispersions.</p><p>Conclusions</p><p>We have characterized a rodent model of IUGR and have demonstrated that the neuroanatomical and neurobehavioural deficits mirror the severity of the IUGR injury. This model has the potential to be applied to examine the pathobiology of and potential therapeutic strategies for IUGR-related brain injury. Thus, this work has potential translational relevance for the study of CP.</p></div

IUGR is accompanied by a loss of white matter in the corpus callosum.

<p>Olig2+ OL counts in the corpus callosum revealed a decrease in OLs in mild IUGR (B), and moderate IUGR (C) animals, but not in sham controls (A). This decrease was significantly different in both IUGR groups when compared to sham controls; moderate IUGR animals also displayed significantly reduced counts when compared to mild IUGR animals (D). Both mild and moderate IUGR animals had decreased myelination (as assessed through MAG+ staining) when compared to sham controls (F-H). The number of NF200+ axons was also reduced in both mild and moderate IUGR groups when compared to sham controls (I-K). Merged images of MAG+/NF200+ staining are also shown (L-N). There were fewer axons in IUGR animals regardless of the severity of injury (O). A significantly greater proportion of the remaining axons were unmyelinated (MAG-/NF200+) in only moderate IUGR animals, while mild IUGR animals exhibited a phenotype similar to sham controls (P). When we investigated the results in (P) as a percentage of total axons in the CC, only moderately injured animals had a significantly higher proportion of unmyelinated axons when compared to other groups (Q). Scale Bar = 100 μm;*p<0.05; **p<0.01; ***p<0.001.</p