Neonatal Neurons Research Articles

MiR-322-5p Alleviates Cell Injury and Impairment of Cognitive Function in Vascular Dementia by Targeting TSPAN5

PurposeAs the population ages, the incidence of clinical dementia has been rising around the world. It has been reported that microRNAs act as key diagnostic biomarkers and targets for various neurological conditions, including dementia. MiR-322-5p has been revealed to play an important role in multiple diseases. In this study, we aimed to investigate the role and regulatory mechanism of miR-322-5p in vascular dementia.Materials and MethodsIn this study, neonatal rat neurons (NRNs) were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to induce cell injury. The animals were subjected to permanent bilateral occlusion of the carotid arteries (2-vessel occlusion, 2VO) to induce the model of chronic brain hypoperfusion.ResultsMiR-322-5p expression was significantly downregulated in the neurons exposed to OGD/R and the hippocampi of 2VO rats. Overexpression of miR-322-5p ameliorated cell apoptosis and the inflammatory response in vitro. In a mechanistic study, miR-322-5p was confirmed to directly target and negatively regulate tetraspanin 5 (TSPAN5) in cultured NRNs. Moreover, overexpression of TSPAN5 could counteract the effects of miR-322-5p overexpression on cell apoptosis and the inflammatory response in OGD/R-treated neurons. More importantly, miR-322-5p improved cognitive ability and inhibited inflammatory production in 2VO rats.ConclusionOverall, the results suggest that miR-322-5p alleviates vascular dementia development by targeting TSPAN5. This discovery may provide a potential therapeutic target for dementia.

MiRNA-384-5p Targets GABRB1 to Regulate Ketamine-Induced Neurotoxicity in Neurons.

To determine the function of miR-384-5p in ketamine-induced neurotoxicity. Neonatal hippocampal neurons were isolated from rats and treated with varying doses of ketamine. RT-qPCR was utilized to measure the miR-384-5p level in ketamine-treated neurons. Neuronal viability was evaluated by MTT assay. TUNEL staining and flow cytometry were applied to measure neuronal apoptosis. H2-DCFDA staining was utilized to detect the intracellular ROS level. Protein levels were measured using Western blotting. A luciferase reporter experiment was used in HEK293T cells to verify the interaction of miR-384-5p with GABRB1. Ketamine induced neurotoxicity and miR-384-5p upregulation in hippocampal neurons. miR-384-5p downregulation mitigated ketamine-induced neurotoxicity by restraining apoptosis and ROS activity in neurons. GABRB1 was demonstrated to be targeted by miR-384-5p. GABRB1 depletion worsened ketamine-induced neurotoxicity. Moreover, GABRB1 depletion lessened the protective effect of miR-384-5p inhibition against ketamine-mediated neurotoxicity. miR-384-5p regulates ketamine-induced neurotoxicity in hippocampal neurons by targeting GABRB1.

Effects of Dexmedetomidine on the Expression of Cytochrome C Oxidase in Neonatal Mouse Primary Hippocampal Neurons Cultured in Propofol

To observe the effects of propofol and dexmedetomidine on the expression of cytochrome c oxidase in primary hippocampal neurons isolated from neonatal mice. C57 neonatal mice were sacrificed within 24 h of birth and hippocampal tissue was collected. Primary hippocampal neurons were cultured in vitro and then randomly divided into a control group (0 μM propofol and 0 μM dexmedetomidine), propofol group (20 μM propofol and 0 μM dexmedetomidine) and propofol+dexmedetomidine group (20 μM propofol and 10 μM dexmedetomidine). The neurons were incubated for 1 h. Neuron morphology was observed under a light microscope. The expression of cytochrome c protein was detected semiquantitatively by Western blot and immunofluorescence staining. Compared with those in the control group, the primary hippocampal neurons in the propofol group were morphologically degenerated, with sparse axons and dendrites; the expression of cytochrome c protein was significantly higher in the propofol group (p<0.0001). The morphology of primary hippocampal neurons in the propofol+dexmedetomidine group was similar to that of primary hippocampal neurons in the control group, with plump and bright cell bodies and thickened neurites intertwined in a neuron network; additionally, cytochrome c protein expression was significantly lower in the hippocampal neurons in the propofol+dexmedetomidine group than in the hippocampal neurons in propofol group (p<0.0001). Dexmedetomidine reversed propofol induced injury and morphological abnormalities of primary hippocampal neurons. The mechanism may be related to the downregulation of cytochrome c protein expression.

Effects of lentivirus-mediated RNA interference of HIF-1α and PTEN on oxygen-glucose deprivation injury in primary cultured rat neurons

To observe the effects of lentivirus-mediated RNA interference (RNAi) of hypoxia-inducible factor 1α (HIF-1α) and phosphatase and tensin homolog on chromosome ten (PTEN) on oxygen-glucose deprivation (OGD) injury in primary cultured rat neurons. Primary cultures of neonatal SD rat neurons were infected by lentiviral vectors carrying short hairpin RNA (shRNA) targeting HIF-1α or PTEN followed 4 days later by hypoxic exposure, and the control neurons were infected with the empty virus only with or without subsequent hypoxic exposure. Twenty-four hours after hypoxia, the interference efficiency was assessed with qRT-PCR, and lactate dehydrogenase (LDH) assay and AnnexinV-FITC/ PI assay were performed to detect neuronal damage and apoptosis. The expressions of the related proteins were determined with Western blotting. Lentivirus-mediated RNAi effectively silenced the mRNA expression of the target genes. HIF-1α silencing obviously aggravated the hypoxia-induced damage and apoptosis of the neurons, enhanced the expression of PTEN protein and significantly lowered the expressions of p-PTEN, p-AKT, NR2A and VEGFa (P < 0.05). PTEN silencing significantly alleviated hypoxia-induced damage and apoptosis of the neurons and increased the cellular expressions of p-PTEN and p-AKT (P < 0.05) without obviously affecting the expressions of HIF-1α, NR2A or VEGFa (P>0.05). An up-regulated expression of HIF-1α causes down-regulation of PTEN expression to protect primary cultured rat neurons against OGD injury.

Neuronal Coherence Agent for Shared Intentionality: A Hypothesis of Neurobiological Processes Occurring during Social Interaction

The present interdisciplinary study discusses the physical foundations of the neurobiological processes occurring during social interaction. The review of the literature establishes the difference between Intentionality and Intention, thereby proposing the theoretical basis of Shared Intentionality in humans. According to the present study, Shared Intentionality in humans (Goal-directed coherence of biological systems), which is the ability among social organisms to instantly select just one stimulus for the entire group, is the outcome of evolutionary development. Therefore, this interaction modality should be the preferred, archetypal, and most propagated modality in organisms, attributed to the Model of Hierarchical Complexity Stage 3. This characteristic of biological systems facilitates the training of the new members of the group and also ensures efficient cooperation among the members of the group without requiring communication. In humans, Shared Intentionality contributes to the learning of newborns. The neurons of a mature organism may teach the neonate neurons regarding the fitting reactions to the excitatory inputs of the specific structural organization. This enables the neonate neurons to develop a Long-Term Potentiation that links particular stimuli with specific embodied sensorimotor neural networks. The present report discusses three possible neuronal coherence agents that could involve quantum mechanisms in cells, thereby enabling the distribution of the quality of goal-directed coherence in biological systems (Shared Intentionality in humans). Recently reported case studies conducted online with the task of conveying the meaning of numerosity to the children of age 18–33 months revealed the occurrence of Shared Intentionality in mother-child dyads in the absence of sensory cues between the two, which promoted cognitive development in the children. The findings of these case studies support the concept of physical foundations and the hypothesis of the neurophysiological process of social interaction proposed in the present study.

Broad Influence of Mutant Ataxin-3 on the Proteome of the Adult Brain, Young Neurons, and Axons Reveals Central Molecular Processes and Biomarkers in SCA3/MJD Using Knock-In Mouse Model.

Spinocerebellar ataxia type 3 (SCA3/MJD) is caused by CAG expansion mutation resulting in a long polyQ domain in mutant ataxin-3. The mutant protein is a special type of protease, deubiquitinase, which may indicate its prominent impact on the regulation of cellular proteins levels and activity. Yet, the global model picture of SCA3 disease progression on the protein level, molecular pathways in the brain, and neurons, is largely unknown. Here, we investigated the molecular SCA3 mechanism using an interdisciplinary research paradigm combining behavioral and molecular aspects of SCA3 in the knock-in ki91 model. We used the behavior, brain magnetic resonance imaging (MRI) and brain tissue examination to correlate the disease stages with brain proteomics, precise axonal proteomics, neuronal energy recordings, and labeling of vesicles. We have demonstrated that altered metabolic and mitochondrial proteins in the brain and the lack of weight gain in Ki91 SCA3/MJD mice is reflected by the failure of energy metabolism recorded in neonatal SCA3 cerebellar neurons. We have determined that further, during disease progression, proteins responsible for metabolism, cytoskeletal architecture, vesicular, and axonal transport are disturbed, revealing axons as one of the essential cell compartments in SCA3 pathogenesis. Therefore we focus on SCA3 pathogenesis in axonal and somatodendritic compartments revealing highly increased axonal localization of protein synthesis machinery, including ribosomes, translation factors, and RNA binding proteins, while the level of proteins responsible for cellular transport and mitochondria was decreased. We demonstrate the accumulation of axonal vesicles in neonatal SCA3 cerebellar neurons and increased phosphorylation of SMI-312 positive adult cerebellar axons, which indicate axonal dysfunction in SCA3. In summary, the SCA3 disease mechanism is based on the broad influence of mutant ataxin-3 on the neuronal proteome. Processes central in our SCA3 model include disturbed localization of proteins between axonal and somatodendritic compartment, early neuronal energy deficit, altered neuronal cytoskeletal structure, an overabundance of various components of protein synthesis machinery in axons.

Orexin-A inhibits γ-aminobutyric acid current of neonatal rat spinal cord ventral horn neurons by activating OX1R, OX2R and Ca2+-independent PKC

To investigate the effect of orexin-A on the functionality of ionotropic γ-aminobutyric acid (GABA) receptors in spinal cord ventral horn neurons and its mechanisms. The spinal cord containing the lumbosacral enlargement was isolated from neonatal SD rats (7-12 days old) and sliced. The slices were digested with papain (in 0.18 g/30 mL artificial cerebrospinal fluid) for 40-60 min, and the ventral horn neurons were separated acutely using fire-polished Pasteur pipettes. After the cells adhered to the bottom of Petri dishes, patch-clamp experiments combined with pharmacological methods were performed to test the effects of orexin-A on GABA currents of the neurons treated with SB334867 (a selective OX1R antagonist), TCSOX229 (a selective OX2R antagonist), Bis-Ⅳ (a PKC inhibitor), PMA (a PKC agonist), Rp-cAMP (a PKA inhibitor), or BAPTA (Ca2+ chelator). The isolated neurons maintained good morphologies with diverse shapes of cell body and long protrusions. Treatment with orexin-A significantly inhibited the amplitude of GABA-induced current (P < 0.001, n=49) with an inhibition rate of (67.48±12.50)%. SB334867 and TCSOX229, when applied simultaneously, completely abolished the suppressive effect of orexin-A on the GABA currents (P=0.93, n=6), and their separate use partially relieved the suppressive effect of orexin-A (P=0.001, n=8; P=0.02, n=8). The application of Bis-Ⅳ also abolished the suppressive effect of orexin-A on GABA currents (P=0.31, n=5). PMA mimicked the effect of orexin-A in these neurons and significantly inhibited GABA currents with an inhibition rate of (60.79±10.94)%, and the application of orexin-A did not cause further suppression of GABA currents in PMA-treated neurons (P=0.15, n=6). Orexin-A was still capable of suppressing GABA currents in Rp-cAMP-treated neurons (P=0.001, n=5). The extracellular Ca2+-free solution (P=0.004, n=8) or the presence of BAPTA (P=0.04, n=7) did not significantly affect the inhibitory effect of orexin-A on GABA currents. Orexin-A inhibits GABA currents in the ventral horn neurons of rat spinal cord probably by activating OX1R, OX2R and Ca2+-independent PKC.

RIPK1/RIPK3-Mediated Necroptosis is Involved in Sevoflurane-Induced Neonatal Neurotoxicity in the Rat Hippocampus.

Recent studies have shown that exposure to sevoflurane in developing brains causes neuronal apoptosis and cognitive dysfunction. "Necroptosis" is a novel pathway of necrosis. We introduced the caspase-specific inhibitor Z-VAD in addition to the receptor-interacting protein kinase 1 (RIPK1) inhibitor Nec-1, to ascertain the existence and importance of necroptosis. Sprague-Dawley rat pups postnatal day 7 were randomly assigned into one of five groups: control, sevoflurane + Z-VAD, sevoflurane + Nec-1, sevoflurane + Z-VAD + Nec-1 and 3% sevoflurane group. Neuronal apoptosis was evaluated by hematoxylin and eosin staining. The MTT assay was performed to evaluate cell viability. Immunofluorescence was employed to measure expression of RIPK1 and RIPK3. Western blots showing expression of RIPK1, RIPK3 and phosphorylation of mixed lineage kinase domain-like (p-MLKL) were used to explore the role of necroptosis. Binding of RIPK1/RIPK3 was detected via co-immunoprecipitation. Finally, the Morris water maze test was used to determine cognitive function. Exposure to 3% sevoflurane for 6h induced neurotoxicity and inhibited cell viability. Neuron viability was low in the SEV, SEV + Z-VAD and SEV + Nec-1 groups. The study revealed that RIPK1 and RIPK3 protein expression increased significantly, but there was no significant differences between the SEV and SEV + Z-VAD groups. The expression of p-MLKL significantly increased in the SEV and SEV + Z-VAD groups, but not in the SEV + Nec-1 group or SEV + Z-VAD + Nec-1 group compared to the control group. Co-immunoprecipitation results showed that sevoflurane exposure enhanced binding of RIPK1/RIPK3 protein significantly. Blockade of apoptosis and necroptosis alleviated sevoflurane-induced cognitive impairment. Sevoflurane exposure elicited neurotoxicity within neonatal hippocampal neurons and tissues. Blockade of apoptosis or necroptosis alone did not attenuate sevoflurane-induced neurotoxicity (SIN). RIPK1/RIPK3-mediated necroptosis was involved in SIN in hippocampal neurons. SIN could be attenuated only by inhibiting both apoptosis and necroptosis.

Melatonin protects inner retinal neurons of newborn mice after hypoxia-ischemia.

Retinopathy of prematurity is a vision-threatening disease associated with retinal hypoxia-ischemia, leading to the death of retinal neurons and chronic neuronal degeneration. During this study, we used the oxygen-induced retinopathy mice model to mimic retinal hypoxia-ischemia phenotypes to investigate further the neuroprotective effect of melatonin on neonatal retinal neurons. Melatonin helped maintain relatively normal inner retinal architecture and thickness and preserve inner retinal neuron populations in avascular areas by rescuing retinal ganglion and bipolar cells, and horizontal and amacrine neurons, from apoptosis. Meanwhile, melatonin recovered visual dysfunction, as reflected by the improved amplitudes and implicit times of a-wave, b-wave, and oscillatory potentials. Additionally, elevated cleaved caspase-3 and Bax protein levels and reduced Bcl-2 protein levels in response to hypoxia-ischemia were diminished after melatonin treatment. Moreover, melatonin increased BDNF and downstream phospho-TrkB/Akt/ERK/CREB levels. ANA-12, a TrkB receptor antagonist, antagonized these melatonin actions and reduced melatonin-induced neuroprotection. Furthermore, melatonin rescued the reduction in melatonin receptor expression. This study suggests that melatonin exerted anti-apoptotic and neuroprotective effects in inner retinal neurons after hypoxia-ischemia, at least partly due to modulation of the BDNF-TrkB pathway.

A5 noradrenergic neurons and breathing control in neonate rats.

The pontine A5 noradrenergic group contributes to the maturation of the respiratory system before birth in rats. These neurons are connected to the neural network responsible for respiratory rhythmogenesis. In the present study, we investigated the participation of A5 noradrenergic neurons in neonates (P7-8 and P14-15) in the control of ventilation during hypoxia and hypercapnia in in vivo experiments using conjugated saporin anti-dopamine beta-hydroxylase (DβH-SAP) to specifically ablate noradrenergic neurons. Thus, DβH-SAP (420ng/μL) or saporin (SAP, control) was injected into the A5 region of neonatal male Wistar rats. Hypoxia reduced respiratory variability in control animals; however, A5 lesion prevented this effect in P7-8 rats. Our data suggest that noradrenergic neurons of the A5 region in neonate rats do not participate in the control of ventilation under baseline and hypercapnic conditions, but exert an inhibitory modulation on breathing variability under hypoxic challenge in early life (P7-8).

MiR-384-3p aggravates propofol induced apoptosis in developing neurons by targeting Wnt3a

Purpose: To evaluate the cytotoxicity of miR-384-3p toward propofol-treated neonatal rat hippocampal neurons and investigate its related molecular mechanism(s).Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine miR-384-3p expression levels in propofol-treated neonatal rat hippocampal neurons. Cell apoptosis and cell viability were evaluated by flow cytometry analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. Bioinformatics and dual-luciferase reporterassay were applied together to forecast and verify the interaction between miR-384-3p and Wnt3a. Wnt3a expression in transfected neonatal rat hippocampal neurons was assessed by Western blot assay.Results: Propofol induced apoptosis in developing neurons by upregulating miR-384-3p expression levels. Knockdown of miR-384-3p reduced propofol-induced apoptosis in developing neurons. Subsequent experiments indicated that miR-384-3p directly regulated Wnt3a expression via coupling with the 3′-untranslated region of Wnt3a. Furthermore, upregulation of Wnt3a expression levels alleviated propofol-induced cytotoxicity promoted by miR-384-3p (p &lt; 0.01).Conclusion: MiR-384-3p aggravates propofol-induced apoptosis in developing neurons by targeting Wnt3a.&#x0D; Keywords: MiR-384-3p, Wnt3a, Propofol, Apoptosis, Developing neurons

Neuregulin-4 Is Required for Maintaining Soma Size of Pyramidal Neurons in the Motor Cortex.

The regulation of neuronal soma size is essential for appropriate brain circuit function and its dysregulation is associated with several neurodevelopmental disorders. A defect in the dendritic growth and elaboration of motor neocortical pyramidal neurons in neonates lacking neuregulin-4 (NRG4) has previously been reported. In this study, we investigated whether the loss of NRG4 causes further morphologic defects that are specific to these neurons. We analyzed the soma size of pyramidal neurons of layer (L)2/3 and L5 of the motor cortex and a subpopulation of multipolar interneurons in this neocortical region in Nrg4+/+ and Nrg4−/− mice. There were significant decreases in pyramidal neuron soma size in Nrg4−/− mice compared with Nrg4+/+ littermates at all stages studied [postnatal day (P)10, P30, and P60]. The reduction was especially marked at P10 and in L5 pyramidal neurons. Soma size was not significantly different for multipolar interneurons at any age. This in vivo phenotype was replicated in pyramidal neurons cultured from Nrg4−/− mice and was rescued by NRG treatment. Analysis of a public single-cell RNA sequencing repository revealed discrete Nrg4 and Erbb4 expression in subpopulations of L5 pyramidal neurons, suggesting that the observed defects were due in part to loss of autocrine Nrg4/ErbB4 signaling. The pyramidal phenotype in the motor cortex of Nrg4−/− mice was associated with a lack of Rotarod test improvement in P60 mice, suggesting that absence of NRG4 causes alterations in motor performance.

Membrane excitabilities in neonatal rat mesencephalic trigeminal neurons under dietary zinc deficiency.

The present study aimed to evaluate the effects of zinc deprivation on the properties of membrane and spike-discharge features of mesencephalic trigeminal neurons (MTNs), which are important sensory neurons for oral-motor reflexes and rhythmical jaw movements. Neonatal Sprague Dawley rats (P10-12) were distributed equally into a normal diet group and a zinc-deficient diet (ZD) group. Whole cell patch-clamp recordings were obtained from MTNs from coronal brain slices. Passive membrane properties showed a modest depolarized membrane potential and decreased cell capacitance in the ZD group. Zinc deprivation decreased the minimal current amplitude, which induced an action potential and increased the amplitude of afterhyperpolarization following the action potential. Negligible changes were observed for other action potential properties. A decreased burst duration was observed, accompanied by hastened spike frequency adaptation in the burst discharge. There was no difference in the resonant properties at both the subthreshold depolarized potential and hyperpolarized membrane potential between the control and ZD groups. These results suggests that neither the persistent sodium conductance nor slow inwardly rectifying conductance were altered; however, there appeared to be an increase in Ca2+-dependent K+ conductance in zincdeficient MTNs.

Postnatal expression and possible function of RANK and RANKL in the murine inner ear.

The bone encasing the inner ear, known as the otic capsule, is unique because it remodels little postnatally compared to other bones in the body. Previous studies established that osteoprotegerin (OPG) in the inner ear inhibits otic capsule remodeling. OPG acts as a decoy receptor of receptor activator of nuclear factor κB ligand (RANKL) to disrupt the interaction between RANKL and RANK, the primary regulators of bone metabolism. Here we studied the expression and function of RANK and RANKL in the murine cochlea. Using a combination of in situ hybridization, real-time quantitative RT-PCR, and western blot, we demonstrate that Rankl and Rank genes and their protein products are expressed in the intracochlear soft tissues and the otic capsule in a developmentally regulated manner. Using a culture of neonatal murine cochlear neurons, we show that the interaction between RANK and RANKL inhibits neurite outgrowth in these neurons, and is associated with upregulation of NOGO-A expression. Taken together, our results suggest that, in addition to regulating otic capsule bone remodeling, RANK and RANKL expressed by intracochlear soft tissues may also regulate spiral ganglion neuron function by affecting neurite outgrowth.

AT1R/GSK-3β/mTOR Signaling Pathway Involved in Angiotensin II-Induced Neuronal Apoptosis after HIE Both In Vitro and In Vivo.

Objective The focus of the present study is to evaluate the effects of Angiotensin II (Ang II) on neuronal apoptosis after HIE and the potential underlying mechanisms. Methods Primary neonatal rat cortical neurons were used to study the oxygen-glucose deprivation (OGD) cell model. The expressions of Ang II, AT1R, GSK-3β, p-GSK-3β, mTOR, p-mTOR, Bax, Bcl-2, and cleaved caspase-3 were detected via western blot. IF and flow cytometry were used to evaluate neuronal apoptosis. Hypoxic-ischemic encephalopathy (HIE) was established to evaluate the therapeutic effects of Ang II in vivo. Cerebral infarction areas were detected by 2,3,5-Triphenyltetrazolium chloride staining. The righting and geotaxis reflexes were also recorded. In addition, Fluoro-Jade C staining and TUNEL staining were performed to evaluate neuronal degeneration and apoptosis. Results Ang II significantly increased the rate of neuronal apoptosis, upregulated the expression of cleaved caspase-3, and downregulated Bcl-2/Bax ratio after OGD insult. For vivo assay, the expressions of endogenous Ang II and AT1R gradually increased and peaked at 24 h after HIE. Ang II increased NeuN-positive AT1R cell expression. In addition, Ang II increased the area of cerebral infarction, promoted neuronal degeneration and apoptosis, aggravated neurological deficits on righting and geotaxis reflexes, and was accompanied by increased expressions of phosphorylated GSK-3β and mTOR. The application of valsartan (Ang II inhibitor) or SB216763 (GSK-3β inhibitor) reversed these phenomena triggered by Ang II following HIE. Conclusion Ang II increased neuronal apoptosis through the AT1R/GSK-3β/mTOR signaling pathway after experimental HIE both in vitro and in vivo, and Ang II may serve as a novel therapeutic target to ameliorate brain injury after HIE.

Overshooting Subcellular Redox-Responses in Rett-Mouse Hippocampus during Neurotransmitter Stimulation.

Rett syndrome (RTT) is a neurodevelopmental disorder associated with disturbed neuronal responsiveness and impaired neuronal network function. Furthermore, mitochondrial alterations and a weakened cellular redox-homeostasis are considered part of the complex pathogenesis. So far, overshooting redox-responses of MeCP2-deficient neurons were observed during oxidant-mediated stress, hypoxia and mitochondrial inhibition. To further clarify the relevance of the fragile redox-balance for the neuronal (dys)function in RTT, we addressed more physiological stimuli and quantified the subcellular redox responses to neurotransmitter-stimulation. The roGFP redox sensor was expressed in either the cytosol or the mitochondrial matrix of cultured mouse hippocampal neurons, and the responses to transient stimulation by glutamate, serotonin, dopamine and norepinephrine were characterized. Each neurotransmitter evoked more intense oxidizing responses in the cytosol of MeCP2-deficient than in wildtype neurons. In the mitochondrial matrix the neurotransmitter-evoked oxidizing changes were more moderate and more uniform among genotypes. This identifies the cytosol as an important reactive oxygen species (ROS) source and as less stably redox buffered. Fura-2 imaging and extracellular Ca2+ withdrawal confirmed cytosolic Ca2+ transients as a contributing factor of neurotransmitter-induced redox responses and their potentiation in the cytosol of MeCP2-deficient neurons. Chemical uncoupling demonstrated the involvement of mitochondria. Nevertheless, cytosolic NADPH- and xanthine oxidases interact to play the leading role in the neurotransmitter-mediated oxidizing responses. As exaggerated redox-responses were already evident in neonatal MeCP2-deficient neurons, they may contribute remarkably to the altered neuronal network performance and the disturbed neuronal signaling, which are among the hallmarks of RTT.

Regulation of degenerative spheroids after injury

Neuronal injury leads to rapid, programmed disintegration of axons distal to the site of lesion. Much like other forms of axon degeneration (e.g. developmental pruning, toxic insult from neurodegenerative disorder), Wallerian degeneration associated with injury is preceded by spheroid formation along axons. The mechanisms by which injury leads to formation of spheroids and whether these spheroids have a functional role in degeneration remain elusive. Here, using neonatal mouse primary sympathetic neurons, we investigate the roles of players previously implicated in the progression of Wallerian degeneration in injury-induced spheroid formation. We find that intra-axonal calcium flux is accompanied by actin-Rho dependent growth of calcium rich axonal spheroids that eventually rupture, releasing material to the extracellular space prior to catastrophic axon degeneration. Importantly, after injury, Sarm1−/− and DR6−/−, but not Wlds (excess NAD+) neurons, are capable of forming spheroids that eventually rupture, releasing their contents to the extracellular space to promote degeneration. Supplementation of exogenous NAD+ or expressing WLDs suppresses Rho-dependent spheroid formation and degeneration in response to injury. Moreover, injured or trophically deprived Sarm1−/− and DR6−/−, but not Wlds neurons, are resistant to degeneration induced by conditioned media collected from wild-type axons after spheroid rupture. Taken together, these findings place Rho-actin and NAD+ upstream of spheroid formation and may suggest that other mediators of degeneration, such as DR6 and SARM1, mediate post-spheroid rupture events that lead to catastrophic axon disassembly.

Abstract MP45: Prorenin Induces Intracellular Signaling And Reactive Oxygen Species In The Brainstem

The activation of the brain renin angiotensin system (RAS) is required for the blood pressure (BP) elevation in models of neurogenic hypertension (HT). However, it remains unclear whether the expression of prorenin and its binding to prorenin receptor (PRR) in particular brain regions is required for the activation of the brain RAS. Compelling new evidence indicates that renin is expressed in the proximity of the rostral ventrolateral medulla (RVLM) and the nucleus ambiguous within the brainstem where RAS genes including PRR, angiotensinogen, and angiotensin receptors were also detected. Thus, we hypothesized that prorenin acts within the brainstem to induce extracellular signal-regulated kinases (ERK1/2) phosphorylation and generation of reactive oxygen species (ROS) resulting in BP elevation through a PRR-dependent mechanism. Neonatal brainstem neurons obtained from mice at postnatal day 1-3 were cultured and subsequently incubated in presence of recombinant prorenin (rProrenin; 100 nM) or vehicle. rProrenin increased the ratio of phosphorylated-to-total ERK1/2 from 0.7±0.1 A.U. at baseline to 1.0±0.1 A.U. at 30 min (54% increase; p=0.004; n=7) and 1.3±0.1 A.U. at 60 min (102% increase; p&lt;0.0001; n=7). Both PRO20, a PRR blocker, and Ro-31, a protein kinase C inhibitor, abrogated prorenin-induced ERK1/2 phosphorylation, suggesting a PRR-dependent component to this response. Treatment with rProrenin increased NADPH oxidase activity by 47.2±9.5 % compared to vehicle control (p&lt;0.05, n=5). RVLM-targeted stereotactic microinjection of glutamate, rProrenin, or Ang II, but not vehicle, induced an acute BP elevation in isoflurane-anesthetized C57BL/6J mice. Moreover, preliminary data indicate that RVLM-targeted ablation of PRR in PRR-flox mice attenuates the pressor response to deoxycorticosterone (DOCA)-salt in females (WT=132±3 vs KO=120±3 mmHg; p&lt;0.05; n=3-4), but not males (n=5-8), during the first week on DOCA-salt treatment. There was no difference in BP during the 2 nd and 3 rd week of DOCA-salt. We conclude that prorenin induces PRR-mediated downstream signaling involving ERK1/2 phosphorylation and generation of ROS in brainstem neurons, which might contribute to BP elevation in neurogenic HT in sex-dependent manner.

MicroRNA-195 prevents hippocampal microglial/macrophage polarization towards the M1 phenotype induced by chronic brain hypoperfusion through regulating CX3CL1/CX3CR1 signaling

BackgroundMicroglial polarization is a dynamic response to acute brain hypoxia induced by stroke and traumatic brain injury (TBI). However, studies on the polarization of microglia in chronic cerebral circulation insufficiency (CCCI) are limited. Our objective was to investigate the effect of CCCI on microglial polarization after chronic brain hypoperfusion (CBH) and explore the underlying molecular mechanisms.MethodsCBH model was established by bilateral common carotid artery occlusion (2-vessel occlusion, 2VO) in rats. Using the stereotaxic injection technique, lenti-pre-miR-195 and anti-miR-195 oligonucleotide fragments (lenti-pre-AMO-miR-195) were injeted into the CA1 region of the hippocampus to construct animal models with high or low expression of miR-195. Immunofluorescence staining and flow cytometry were conducted to examine the status of microglial polarization. In vitro, Transwell co-culture system was taken to investigate the role of miR-195 on neuronal-microglial communication through CX3CL1-CX3CR1 signaling. Quantitative real-time PCR was used to detect the level of miR-195 and inflammatory factors. The protein levels of CX3CL1 and CX3CR1 were evaluated by both western blot and immunofluorescence staining.ResultsCBH induced by 2VO initiated microglial/macrophage activation in the rat hippocampus from 1 week to 8 weeks, as evaluated by increased ratio of (CD68+ and CD206+)/Iba-1 immunofluorescence. And the microglial/macrophage polarization was shifted towards the M1 phenotype at 8 weeks following CBH. The expression of CX3CL1 and CX3CR1 was increased in the hippocampus of 2VO rats at 8 weeks. An in vitro study in a Transwell co-culture system demonstrated that transfection of either primary-cultured neonatal rat neurons (NRNs) or microglial BV2 cells with AMO-195-induced M1 polarization of BV2 cells and increased CX3CL1 and CX3CR1 expression and that these effects were reversed by miR-195 mimics. Furthermore, the upregulation of miR-195 induced by lenti-pre-miR-195 injection prevented microglial/macrophage polarization to M1 phenotype triggered by hippocampal injection of lenti-pre-AMO-miR-195 and 2VO surgery.ConclusionsOur findings conclude that downregulation of miR-195 in the hippocampus is involved in CBH-induced microglial/macrophage polarization towards M1 phenotype by governing communication between neurons and microglia through the regulation of CX3CL1 and CX3CR1 signaling. This indicates that miR-195 may provide a new strategy for clinical prevention and treatment of CBH.

Gradients in the biophysical properties of neonatal auditory neurons align with synaptic contact position and the intensity coding map of inner hair cells.

Sound intensity is encoded by auditory neuron subgroups that differ in thresholds and spontaneous rates. Whether variations in neuronal biophysics contributes to this functional diversity is unknown. Because intensity thresholds correlate with synaptic position on sensory hair cells, we combined patch clamping with fiber labeling in semi-intact cochlear preparations in neonatal rats from both sexes. The biophysical properties of auditory neurons vary in a striking spatial gradient with synaptic position. Neurons with high thresholds to injected currents contact hair cells at synaptic positions where neurons with high thresholds to sound-intensity are found in vivo. Alignment between in vitro and in vivo thresholds suggests that biophysical variability contributes to intensity coding. Biophysical gradients were evident at all ages examined, indicating that cell diversity emerges in early post-natal development and persists even after continued maturation. This stability enabled a remarkably successful model for predicting synaptic position based solely on biophysical properties.