Mature Hippocampal Neurons Research Articles

Missense mutation in the activation segment of the kinase CK2 models Okur-Chung neurodevelopmental disorder and alters the hippocampal glutamatergic synapse.

Exome sequencing has enabled the identification of causative genes of monogenic forms of autism, amongst them, in 2016, CSNK2A1, the gene encoding the catalytic subunit of the kinase CK2, linking this kinase to Okur-Chung Neurodevelopmental Syndrome (OCNDS), a newly described neurodevelopmental condition with many symptoms resembling those of autism spectrum disorder. Thus far, no preclinical model of this condition exists. Here we describe a knock-in mouse model that harbors the K198R mutation in the activation segment of the α subunit ofCK2. This region is a mutational hotspot, representing one-third of patients. These mice exhibit behavioral phenotypes that mirror patient symptoms. Homozygous knock-in mice die mid-gestation while heterozygous knock-in mice are born at half of the expected mendelian ratio and are smaller in weight and size than wildtype littermates. Heterozygous knock-in mice showed alterations in cognition and memory-assessing paradigms, enhanced stereotypies, altered circadian activity patterns, and nesting behavior. Phosphoproteome analysis from brain tissue revealed alterations in the phosphorylation status of major pre- and postsynaptic proteins of heterozygous knock-in mice. In congruence, we detect reduced synaptic maturation in hippocampal neurons and attenuated long-term potentiation in the hippocampus of knock-in mice. Taken together, heterozygous knock-in mice (CK2αK198R/+) exhibit significant face validity, presenting ASD-relevant phenotypes, synaptic deficits, and alterations in synaptic plasticity, all of which strongly validate this line as a mouse model of OCNDS.

Cyclase-associated protein (CAP) inhibits inverted formin 2 (INF2) to induce dendritic spine maturation

The morphology of dendritic spines, the postsynaptic compartment of most excitatory synapses, decisively modulates the function of neuronal circuits as also evident from human brain disorders associated with altered spine density or morphology. Actin filaments (F-actin) form the backbone of spines, and a number of actin-binding proteins (ABP) have been implicated in shaping the cytoskeleton in mature spines. Instead, only little is known about the mechanisms that control the reorganization from unbranched F-actin of immature spines to the complex, highly branched cytoskeleton of mature spines. Here, we demonstrate impaired spine maturation in hippocampal neurons upon genetic inactivation of cyclase-associated protein 1 (CAP1) and CAP2, but not of CAP1 or CAP2 alone. We found a similar spine maturation defect upon overactivation of inverted formin 2 (INF2), a nucleator of unbranched F-actin with hitherto unknown synaptic function. While INF2 overactivation failed in altering spine density or morphology in CAP-deficient neurons, INF2 inactivation largely rescued their spine defects. From our data we conclude that CAPs inhibit INF2 to induce spine maturation. Since we previously showed that CAPs promote cofilin1-mediated cytoskeletal remodeling in mature spines, we identified them as a molecular switch that control transition from filopodia-like to mature spines.

Mild neonatal hypoxia disrupts adult hippocampal learning and memory and is associated with CK2-mediated dysregulation of synaptic calcium-activated potassium channel KCNN2.

Although nearly half of preterm survivors display persistent neurobehavioral dysfunction including memory impairment without overt gray matter injury, the underlying mechanisms of neuronal or glial dysfunction, and their relationship to commonly observed cerebral white matter injury are unclear. We developed a mouse model to test the hypothesis that mild hypoxia during preterm equivalence is sufficient to persistently disrupt hippocampal neuronal maturation related to adult cellular mechanisms of learning and memory. Methods: Neonatal (P2) mice were exposed to mild hypoxia (8%O 2 ) for 30 min and evaluated for acute injury responses or survived until adulthood for assessment of learning and memory and hippocampal neurodevelopment. Neonatal mild hypoxia resulted in clinically relevant oxygen desaturation and tachycardia without bradycardia and was not accompanied by cerebral gray or white matter injury. Neonatal hypoxia exposure was sufficient to cause hippocampal learning and memory deficits and abnormal maturation of CA1 neurons that persisted into adulthood. This was accompanied by reduced hippocampal CA3-CA1 synaptic strength and LTP and reduced synaptic activity of calcium-sensitive SK2 channels, key regulators of spike timing dependent neuroplasticity, including LTP. Structural illumination microscopy revealed reduced synaptic density, but intact SK2 localization at the synapse. Persistent loss of SK2 activity was mediated by altered casein kinase 2 (CK2) signaling. Clinically relevant mild hypoxic exposure in the neonatal mouse is sufficient to produce morphometric and functional disturbances in hippocampal neuronal maturation independently of white matter injury. Additionally, we describe a novel persistent mechanism of potassium channel dysregulation after neonatal hypoxia. Collectively our findings suggest an unexplored explanation for the broad spectrum of neurobehavioral, cognitive and learning disabilities that paradoxically persist into adulthood without overt gray matter injury after preterm birth.

PHusion - a robust and versatile toolset for automated detection and analysis of exocytosis.

Exocytosis is a fundamental process used by eukaryotes to regulate the composition of the plasma membrane and facilitate cell-cell communication. To investigate exocytosis in neuronal morphogenesis, previously we developed computational tools with a graphical user interface to enable the automatic detection and analysis of exocytic events from fluorescence timelapse images. Although these tools were useful, we found the code was brittle and not easily adapted to different experimental conditions. Here, we developed and validated a robust and versatile toolkit, named pHusion, for the analysis of exocytosis, written in ImageTank, a graphical programming language that combines image visualization and numerical methods. We tested pHusion using a variety of imaging modalities and pH-sensitive fluorophores, diverse cell types and various exocytic markers, to generate a flexible and intuitive package. Using this system, we show that VAMP3-mediated exocytosis occurs 30-times more frequently in melanoma cells compared with primary oligodendrocytes, that VAMP2-mediated fusion events in mature rat hippocampal neurons are longer lasting than those in immature murine cortical neurons, and that exocytic events are clustered in space yet random in time in developing cortical neurons.

Uncovering the Early Events Associated with Oligomeric Aβ-Induced Src Activation.

Soluble Aβ1-42 oligomers (AβO) are formed in the early stages of Alzheimer's disease (AD) and were previously shown to trigger enhanced Ca2+ levels and mitochondrial dysfunction via the activation of N-methyl-D-aspartate receptors (NMDAR). Src kinase is a ubiquitous redox-sensitive non-receptor tyrosine kinase involved in the regulation of several cellular processes, which was demonstrated to have a reciprocal interaction towards NMDAR activation. However, little is known about the early-stage mechanisms associated with AβO-induced neurodysfunction involving Src. Thus, in this work, we analysed the influence of brief exposure to oligomeric Aβ1-42 on Src activation and related mechanisms involving mitochondria and redox changes in mature primary rat hippocampal neurons. Data show that brief exposure to AβO induce H2O2-dependent Src activation involving different cellular events, including NMDAR activation and mediated intracellular Ca2+ rise, enhanced cytosolic and subsequent mitochondrial H2O2 levels, accompanied by mild mitochondrial fragmentation. Interestingly, these effects were prevented by Src inhibition, suggesting a feedforward modulation. The current study supports a relevant role for Src kinase activation in promoting the loss of postsynaptic glutamatergic synapse homeostasis involving cytosolic and mitochondrial ROS generation after brief exposure to AβO. Therefore, restoring Src activity can constitute a protective strategy for mitochondria and related hippocampal glutamatergic synapses.

Cyclosporine A (CsA) prevents synaptic impairment caused by truncated tau by caspase-3

During Alzheimer's (AD), tau protein suffers from abnormal post-translational modifications, including cleaving by caspase-3. These tau forms affect synaptic plasticity contributing to the cognitive decline observed in the early stages of AD. In addition, caspase-3 cleaved tau (TauC3) impairs mitochondrial dynamics and organelles transport, which are both relevant processes for synapse. We recently showed that the absence of tau expression reverts age-associated cognitive and mitochondrial failure by blocking the mitochondrial permeability transition pore (mPTP). mPTP is a mitochondrial complex involved in calcium regulation and apoptosis. Therefore, we studied the effects of TauC3 against the dendritic spine and synaptic vesicle formation and the possible role of mPTP in these alterations. We used mature hippocampal mice neurons to express a reporter protein (GFP, mCherry), coupled to full-length human tau protein (GFP-T4, mCherry-T4), and coupled to human tau protein cleaved at D421 by caspase-3 (GFP-T4C3, mCherry-T4C3) and synaptic elements were evaluated.Treatment with cyclosporine A (CsA), an immunosuppressive drug with inhibitory activity on mPTP, prevented ROS increase and mitochondrial depolarization induced by TauC3 in hippocampal neurons. These results were corroborated with immortalized cortical neurons in which ROS increase and ATP loss induced by this tau form were prevented by CsA. Interestingly, TauC3 expression significantly reduced dendritic spine density (filopodia type) and synaptic vesicle number in hippocampal neurons. Also, neurons transfected with TauC3 showed a significant accumulation of synaptophysin protein in their soma. More importantly, all these synaptic alterations were prevented by CsA, suggesting an mPTP role in these negative changes derived from TauC3 expression.

Neuronal Differentiation and Outgrowth Effect of Thymol in Trachyspermum ammi Seed Extract via BDNF/TrkB Signaling Pathway in Prenatal Maternal Supplementation and Primary Hippocampal Culture.

Reviving the neuronal functions in neurodegenerative disorders requires the promotion of neurite outgrowth. Thymol, which is a principal component of Trachyspermum ammi seed extract (TASE), is reported to have neuroprotective effects. However, the effects of thymol and TASE on neuronal differentiation and outgrowth are yet to be studied. This study is the first report investigating the neuronal growth and maturation effects of TASE and thymol. Pregnant mice were orally supplemented with TASE (250 and 500 mg/kg), thymol (50 and 100 mg/kg), vehicle, and positive controls. The supplementation significantly upregulated the expression of brain-derived neurotrophic factor (BDNF) and early neuritogenesis markers in the pups' brains at post-natal day 1 (P1). Similarly, the BDNF level was significantly upregulated in the P12 pups' brains. Furthermore, TASE (75 and 100 µg/mL) and thymol (10 and 20 µM) enhanced the neuronal polarity, early neurite arborization, and maturation of hippocampal neurons in a dose-dependent manner in primary hippocampal cultures. The stimulatory activities of TASE and thymol on neurite extension involved TrkB signaling, as evidenced by attenuation via ANA-12 (5 µM), which is a specific TrkB inhibitor. Moreover, TASE and thymol rescued the nocodazole-induced blunted neurite extension in primary hippocampal cultures, suggesting their role as a potent microtubule stabilizing agent. These findings demonstrate the potent capacities of TASE and thymol in promoting neuronal development and reconstruction of neuronal circuitry, which are often compromised in neurodegenerative diseases and acute brain injuries.

Sex Significantly Impacts the Function of Major Depression–Linked Variants In Vivo

BackgroundGenome-wide association studies have discovered blocks of common variants—likely transcriptional-regulatory—associated with major depressive disorder (MDD), though the functional subset and their biological impacts remain unknown. Likewise, why depression occurs in females more frequently than males is unclear. We therefore tested the hypothesis that risk-associated functional variants interact with sex and produce greater impact in female brains. MethodsWe developed techniques to directly measure regulatory variant activity and sex interactions using massively parallel reporter assays in the mouse brain in vivo, in a cell type–specific manner, and applied these approaches to measure activity of >1000 variants from >30 MDD loci. ResultsWe identified extensive sex-by-allele effects in mature hippocampal neurons, suggesting that sex-differentiated impacts of genetic risk may underlie sex bias in disease. Unbiased informatics approaches indicated that functional MDD variants recurrently disrupt a number of transcription factor binding motifs, including those of sex hormone receptors. We confirmed a role for the latter by performing massively parallel reporter assays in neonatal mice on the day of birth (during a sex-differentiating hormone surge) and hormonally quiescent juveniles. ConclusionsOur study provides novel insights into the influence of age, biological sex, and cell type on regulatory variant function and provides a framework for in vivo parallel assays to functionally define interactions between organismal variables such as sex and regulatory variation. Moreover, we experimentally demonstrate that a portion of the sex differences seen in MDD occurrence may be a product of sex-differentiated effects at associated regulatory variants.

Lateral Diffusion of NKCC1 Contributes to Chloride Homeostasis in Neurons and Is Rapidly Regulated by the WNK Signaling Pathway.

An upregulation of the Na+-K+-2Cl- cotransporter NKCC1, the main chloride importer in mature neurons, can lead to depolarizing/excitatory responses mediated by GABA type A receptors (GABAARs) and, thus, to hyperactivity. Understanding the regulatory mechanisms of NKCC1 would help prevent intra-neuronal chloride accumulation that occurs in pathologies with defective inhibition. The cell mechanisms regulating NKCC1 are poorly understood. Here, we report in mature hippocampal neurons that GABAergic activity controls the membrane diffusion and clustering of NKCC1 via the chloride-sensitive WNK lysine deficient protein kinase 1 (WNK1) and the downstream Ste20 Pro-line Asparagine Rich Kinase (SPAK) kinase that directly phosphorylates NKCC1 on key threonine residues. At rest, this signaling pathway has little effect on intracellular Cl- concentration, but it participates in the elevation of intraneuronal Cl- concentration in hyperactivity conditions associated with an up-regulation of NKCC1. The fact that the main chloride exporter, the K+-Cl- cotransporter KCC2, is also regulated in mature neurons by the WNK1 pathway indicates that this pathway will be a target of choice in the pathology.

Differentiated HT22 cells as a novel model for in vitro screening of serotonin reuptake inhibitors.

The mouse hippocampal neuronal cell line HT22 is frequently used as an in vitro model to investigate the role of hippocampal cholinergic neurons in cognitive functions. HT22 cells are derived from hippocampal neuronal HT4 cells. However, whether these cells exhibit the serotonergic neuronal phenotype observed in mature hippocampal neurons has not been determined yet. In this present study, we examined whether the differentiation of HT22 cells enhances the serotonergic neuronal phenotype, and if so, whether it can be used for antidepressant screening. Our results show that differentiation of HT22 cells promoted neurite outgrowth and upregulation of N-methyl-D-aspartate receptor and choline acetyltransferase, which is similar to that observed in primary cultured hippocampal neurons. Furthermore, proteins required for serotonergic neurotransmission, such as tryptophan hydroxylase 2, serotonin (5-hydroxytryptamine, 5-HT)1a receptor, and serotonin transporter (SERT), were significantly upregulated in differentiated HT22 cells. The transcription factor Pet-1 was upregulated during HT22 differentiation and was responsible for the regulation of the serotonergic neuronal phenotype. Differentiation also enhanced the functional serotonergic properties of HT22 cells, as evidenced by increase in intracellular 5-HT levels, serotonin transporter SERT glycosylation, and 5-HT reuptake activity. The sensitivity of 5-HT reuptake inhibition by venlafaxine in differentiated HT22 cells (IC50, 27.21nM) was comparable to that in HEK293 cells overexpressing serotonin transporter SERT (IC50, 30.65nM). These findings suggest that the differentiation of HT22 cells enhances their functional serotonergic properties, and these cells could be a potential in vitro system for assessing the efficacy of antidepressant 5-HT reuptake inhibitors.

Riluzole and novel naphthalenyl substituted aminothiazole derivatives prevent acute neural excitotoxic injury in a rat model of temporal lobe epilepsy

Epileptogenic seizures, or status epilepticus (SE), leads to excitotoxic injury in hippocampal and limbic neurons in the kainic acid (KA) animal model of temporal lobe epilepsy (TLE). Here, we have further characterized neural activity regulated methylaminoisobutryic acid (MeAIB)/glutamine transport activity in mature rat hippocampal neurons in vitro that is inhibited by riluzole (IC50 = 1 μM), an anti-convulsant benzothiazole agent. We screened a library of riluzole derivatives and identified SKA-41 followed by a second screen and synthesized several novel chlorinated aminothiazoles (SKA-377, SKA-378, SKA-379) that are also potent MeAIB transport inhibitors in vitro, and brain penetrant following systemic administration. When administered before KA, SKA-378 did not prevent seizures but still protected the hippocampus and several other limbic areas against SE-induced neurodegeneration at 3d. When SKA-377 - 379, (30 mg/kg) were administered after KA-induced SE, acute neural injury in the CA3, CA1 and CA4/hilus was also largely attenuated. Riluzole (10 mg/kg) blocks acute neural injury. Kinetic analysis of SKA-378 and riluzoles’ blockade of Ca2+-regulated MeAIB transport in neurons in vitro indicates that inhibition occurs via a non-competitive, indirect mechanism. Sodium channel NaV1.6 antagonism blocks neural activity regulated MeAIB/Gln transport in vitro (IC50 = 60 nM) and SKA-378 is the most potent inhibitor of NaV1.6 (IC50 = 28 μM) compared to NaV1.2 (IC50 = 118 μM) in heterologous cells. However, pharmacokinetic analysis suggests that sodium channel blockade may not be the predominant mechanism of neuroprotection here. Riluzole and our novel aminothiazoles are agents that attenuate acute neural hippocampal injury following KA-induced SE and may help to understand mechanisms involved in the progression of epileptic disease.

Adenosine A1 receptor antagonist-induced facilitation of postsynaptic AMPA currents in pyramidal neurons of the rat hippocampal CA2 area.

Adenosine A1 receptors (A1R) are widely expressed in hippocampal pyramidal neurons and their presynaptic terminals. It is well known that endogenous adenosine regulates hippocampal function through the activation of A1R in hippocampal pyramidal neurons and has been reported that blockade of A1R induces stronger potentiation of excitatory synaptic transmission in CA2 pyramidal neurons than in CA1 pyramidal neurons. This strong potentiation of CA2 neurons is thought to be caused by the specific modulation of excitatory synaptic transmission through postsynaptic A1R. However, the direct effects of A1R on postsynaptic AMPA channels remain unknown because of the technical difficulties of patch-clamp recording from mature hippocampal CA2 neurons. We recorded synaptic currents from pyramidal neurons in CA1 and CA2 and analyzed the effects of an A1R antagonist on stimulation-evoked synaptic transmission and local application-induced postsynaptic AMPA currents. The antagonist increased the amplitude of evoked synaptic transmission in neurons in both CA1 and CA2. This facilitation was larger in pyramidal neurons in CA2 than in CA1. The antagonist also increased postsynaptic AMPA currents in neurons in CA2 but not in CA1. This facilitation of CA2 AMPA currents was occluded by the intracellular application of a G-protein blocker. Even with the blockade of postsynaptic G-protein signaling, the A1R antagonist increased evoked synaptic transmission in neurons in CA2. These results suggest that synaptic transmission in pyramidal neurons in CA2 is regulated by both presynaptic and postsynaptic A1R. Moreover, A1R regulate excitatory synaptic transmission in pyramidal neurons in CA2 through the characteristic postsynaptic modulation of AMPA currents.

Deciphering the Heterogeneity of the Internal Environment of Hippocampal Neurons during Maturation by Raman Spectroscopy.

Hippocampal neurons are sensitive to changes in the internal environment and play a significant role in controlling learning, memory, and emotions. A remarkable characteristic of the aging brain is its ability to shift from a state of normal inflammation to excessive inflammation. Various cognitive abilities of the elderly may suffer from serious harm due to the change in the neural environment. Hippocampal neurons may have various subsets involved in controlling their internal environment at different stages of development. Developmental differences may eventually result from complex changes in the dynamic neuronal system brought on by metabolic changes. In this study, we used an in vitro hippocampal neuron model cultured in C57BL/6J mice in conjugation with Raman spectroscopy to examine the relative alterations in potential biomarkers, such as levels of metabolites in the internal environment of hippocampal neurons at various developmental stages. The various differentially expressed genes (DEGs) of hippocampal neurons at various developmental stages were simultaneously screened using bioinformatics, and the biological functions as well as the various regulatory pathways of DEGs were preliminarily analyzed, providing an essential reference for investigating novel therapeutic approaches for diseases that cause cognitive impairment, such as Alzheimer’s disease. A stable hippocampal neuron model was established using the GIBCO C57BL/6J hippocampal neuron cell line as a donor and in vitro hippocampal neuron culture technology. The Raman peak intensities of culture supernatants from the experimental groups incubated for 0, 7, and 14 days in vitro(DIV) were examined. The GEO database was used to screen for different DEGs associated with various developmental stages. The data was then analyzed using a statistical method called orthogonal partial least squares discriminant analysis (OPLS-DA). The levels of ketogenic and glycogenic amino acids (such as tryptophan, phenylalanine, and tyrosine), lipid intake rate, glucose utilization rate, and nucleic acid expression in the internal environment of hippocampal neurons were significantly different in the 14 DIV group compared to the 0 DIV and 7 DIV groups (P < 0.01). The top 10 DEGs with neuronal maturation were screened, and the results were compared to the OPLS-DA model’s analysis of the differential peaks. It was found that different genes involved in maturation can directly relate to changes in the body’s levels of ketogenic and glycogenic amino acids (P < 0.01). The altered expression of the maturation-related genes epidermal growth factor receptor, protein tyrosine kinase 2-beta, discs large MAGUK scaffold protein 2, and Ras protein-specific guanine nucleotide releasing factor 1 may be connected to the altered uptake of ketogenic and glycogenic amino acids and nucleic acids in the internal environment of neurons at different developmental stages. The levels of ketogenic, glycogenic amino acids, and lipid intake increased while glucose utilization decreased, which may be related to mature neurons’ metabolism and energy use. The decline in nucleic acid consumption could be connected to synaptic failure. The Raman spectroscopy fingerprint results of relevant biomarkers in conjugation with multivariable analysis and biological action targets suggested by differential genes interpret the heterogeneity of the internal environment of mature hippocampal neurons in the process of maturation, open a new idea for exploring the dynamic mechanism of the exchange energy metabolism of information molecules in the internal environment of hippocampal neurons, and provide a new method for studying this process.

Analysis of the Expression and Subcellular Distribution of eEF1A1 and eEF1A2 mRNAs during Neurodevelopment.

Neurodevelopment is accompanied by a precise change in the expression of the translation elongation factor 1A variants from eEF1A1 to eEF1A2. These are paralogue genes that encode 92% identical proteins in mammals. The switch in the expression of eEF1A variants has been well studied in mouse motor neurons, which solely express eEF1A2 by four weeks of postnatal development. However, changes in the subcellular localization of eEF1A variants during neurodevelopment have not been studied in detail in other neuronal types because antibodies lack perfect specificity, and immunofluorescence has a low sensitivity. In hippocampal neurons, eEF1A is related to synaptic plasticity and memory consolidation, and decreased eEF1A expression is observed in the hippocampus of Alzheimer’s patients. However, the specific variant involved in these functions is unknown. To distinguish eEF1A1 from eEF1A2 expression, we have designed single-molecule fluorescence in-situ hybridization probes to detect either eEF1A1 or eEF1A2 mRNAs in cultured primary hippocampal neurons and brain tissues. We have developed a computational framework, ARLIN (analysis of RNA localization in neurons), to analyze and compare the subcellular distribution of eEF1A1 and eEF1A2 mRNAs at specific developmental stages and in mature neurons. We found that eEF1A1 and eEF1A2 mRNAs differ in expression and subcellular localization over neurodevelopment, and eEF1A1 mRNAs localize in dendrites and synapses during dendritogenesis and synaptogenesis. Interestingly, mature hippocampal neurons coexpress both variant mRNAs, and eEF1A1 remains the predominant variant in dendrites.

Generation and characterization of the adult neuron-specific Aralar/AGC1 knock-out mice.

Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier expressed in neurons, is the regulatory component of the NADH malate-aspartate shuttle. AGC1 deficiency is a neuropediatric rare disease (OMIM #612949, also named early infantile epileptic encephalopathy 39) characterized by hypomyelination, hypotonia, developmental arrest and epilepsy. In mice, the global aralar knockout shows hyperreactivity, growth retardation, motor discoordination, seizures and hypomyelination; thus, recapitulating the pathology of the human disease. Aralar brain expression is mainly restricted to neurons, while glial expression of Aralar has been demonstrated to be scarce. However, Aralar deficiency manifests alteration in fundamental glial functions such as glutamine and myelination synthesis by astrocytes and oligodendrocytes respectively. In order to dissect the role of neuronal Aralar in the disease mechanism of AGC1 deficiency, we have ablated the expression of Aralar in mature (from PND30 onward) excitatory cortical and hippocampal neurons using the CaMKIIα-driven Cre recombinase expression in Aralarlox/lox mice. Here, we present the characterization of the mature neuron-specific aralar knock out mice. We demonstrated that the excision of exon 3 aralar starts from PND30 and continues to occur during mouse adulthood. Consequently, Aralar protein levels gradually decay across the 2nd to 6th month up to a 70- 75% decrease in the cortex and a 50% decrease in the hippocampus. The drop in Aralar levels drive an alteration in cortical metabolites causing a 25% and 50% decrease in aspartate and serine levels, the metabolites most affected in the brain of the global aralar-KO mice. This partial decreases in ARALAR and metabolites caused by cortical CaMKIIα-driven aralar ablation are not accompanied by growth or behavioral alterations in a battery of motor, memory and cognitive tests. The results suggest that the absence of Aralar in CaMKIIα expressing neurons (cortical and hippocampal mature excitatory neurons), does not mimic the alterations caused by global deficiency of Aralar/AGC1. However, since the levels of cortical ARALAR in this model resemble those of healthy aralar+/- mice, further experiments are being carried out to explore the origin (or location) of the ARALAR protein remnants and the possible metabolic compensation by other neurons and/or cell types in these mice. Without ruling out either that the ARALAR-MAS pathway could play a more critical role during neurodevelopment than adulthood in brain physiology and metabolism.

A role for insulin-like growth factor-1 in hippocampal plasticity following traumatic brain injury.

Traumatic brain injury (TBI) initiates a constellation of secondary injury cascades, leading to neuronal damage and dysfunction that is often beyond the scope of endogenous repair mechanisms. Cognitive deficits are among the most persistent morbidities resulting from TBI, necessitating a greater understanding of mechanisms of posttraumatic hippocampal damage and neuroplasticity and identification of therapies that improve recovery by enhancing repair pathways. Focusing here on hippocampal neuropathology associated with contusion-type TBIs, the impact of brain trauma on synaptic structure and function and the process of adult neurogenesis is discussed, reviewing initial patterns of damage as well as evidence for spontaneous recovery. A case is made that insulin-like growth factor-1 (IGF-1), a growth-promoting peptide synthesized in both the brain and the periphery, is well suited to augment neuroplasticity in the injured brain. Essential during brain development, multiple lines of evidence delineate roles in the adult brain for IGF-1 in the maintenance of synapses, regulation of neurotransmission, and modulation of forms of synaptic plasticity such as long-term potentiation. Further, IGF-1 enhances adult hippocampal neurogenesis though effects on proliferation and neuronal differentiation of neural progenitor cells and on dendritic growth of newly born neurons. Post-injury administration of IGF-1 has been effective in rodent models of TBI in improving learning and memory, attenuating death of mature hippocampal neurons and promoting neurogenesis, providing critical proof-of-concept data. More studies are needed to explore the effects of IGF-1-based therapies on synaptogenesis and synaptic plasticity following TBI and to optimize strategies in order to stimulate only appropriate, functional neuroplasticity.

Astrocytic ApoE underlies maturation of hippocampal neurons and cognitive recovery after traumatic brain injury in mice

Polymorphisms in the apolipoprotein E (ApoE) gene confer a major genetic risk for the development of late-onset Alzheimer’s disease (AD) and are predictive of outcome following traumatic brain injury (TBI). Alterations in adult hippocampal neurogenesis have long been associated with both the development of AD and recovery following TBI and ApoE is known to play a role in this process. In order to determine how ApoE might influence hippocampal injury-induced neurogenesis, we generated a conditional knockout system whereby functional ApoE from astrocytes was ablated prior to injury. While successfully ablating ApoE just prior to TBI in mice, we observed an attenuation in the development of the spines in the newborn neurons. Intriguingly, animals with a double-hit, i.e. injury and ApoE conditionally inactivated in astrocytes, demonstrated the most pronounced impairments in the hippocampal-dependent Morris water maze test, failing to exhibit spatial memory after both acquisition and reversal training trials. In comparison, conditional knockout mice without injury displayed impairments but only in the reversal phase of the test, suggesting accumulative effects of astrocytic ApoE deficiency and traumatic brain injury on AD-like phenotypes. Together, these findings demonstrate that astrocytic ApoE is required for functional injury-induced neurogenesis following traumatic brain injury.

Staufen 1 is expressed by neural precursor cells in the developing murine cortex but is dispensable for NPC self-renewal and neuronal differentiation in vitro

BackgroundProper development of the cerebral cortex relies on asymmetric divisions of neural precursor cells (NPCs) to produce a recurring NPC and a differentiated neuron. Asymmetric divisions are promoted by the differential localization of cell-fate determinants, such as mRNA, between daughter cells. Staufen 1 (Stau1) is an RNA-binding protein known to localize mRNA in mature hippocampal neurons. Its expression pattern and role in the developing mammalian cortex remains unknown. ResultsBoth stau1 mRNA and Stau1 protein were found to be expressed in all cells of the developing murine cortex. Stau1 protein expression was characterized spatially and temporally throughout cortical development and found to be present in all stages investigated. We observed expression in the nucleus, cytoplasm and distal processes of both NPCs and newly born neurons and found it to shuttle between the nucleus and the cytoplasm. Upon shRNA-mediated knock-down of Stau1 in primary cultures of the developing cortex, we did not observe any phenotype in NPCs. They were able to both self-renew and generate neurons in the absence of Stau1 expression. ConclusionsWe propose that Stau1 is either dispensable for the development of the cerebral cortex or that its paralogue, Stau2, is able to compensate for its loss.

MiR-9-5p is involved in the rescue of stress-dependent dendritic shortening of hippocampal pyramidal neurons induced by acute antidepressant treatment with ketamine

Converging clinical and preclinical evidence demonstrates that depressive phenotypes are associated with synaptic dysfunction and dendritic simplification in cortico-limbic glutamatergic areas. On the other hand, the rapid antidepressant effect of acute ketamine is consistently reported to occur together with the rescue of dendritic atrophy and reduction of spine number induced by chronic stress in the hippocampus and prefrontal cortex of animal models of depression. Nevertheless, the molecular mechanisms underlying these morphological alterations remain largely unknown.Here, we found that miR-9-5p levels were selectively reduced in the hippocampus of rats vulnerable to Chronic Mild Stress (CMS), while acute subanesthetic ketamine restored its levels to basal condition in just 24h; miR-9-5p expression inversely correlated with the anhedonic phenotype. A decrease of miR-9-5p was reproduced in an in vitro model of stress, based on primary hippocampal neurons incubated with the stress hormone corticosterone. In both CMS animals and primary neurons, decreased miR-9-5p levels were associated with dendritic simplification, while treatment with ketamine completely rescued the changes.In vitro modulation of miR-9-5p expression showed a direct role of miR-9-5p in regulating dendritic length and spine density in mature primary hippocampal neurons. Among the putative target genes tested, Rest and Sirt1 were validated as biological targets in primary neuronal cultures. Moreover, in line with miR-9-5p changes, REST protein expression levels were remarkably increased in both CMS vulnerable animals and corticosterone-treated neurons, while ketamine completely abolished this alteration. Finally, the shortening of dendritic length in corticosterone-treated neurons was shown to be partly rescued by miR-9-5p overexpression and dependent on REST protein expression.Overall, our data unveiled the functional role of miR-9-5p in the remodeling of dendritic arbor induced by stress/corticosterone in vulnerable animals and its rescue by acute antidepressant treatment with ketamine.

MeCP2 gates spatial learning-induced alternative splicing events in the mouse hippocampus

Long-term memory formation is supported by functional and structural changes of neuronal networks, which rely on de novo gene transcription and protein synthesis. The modulation of the neuronal transcriptome in response to learning depends on transcriptional and post-transcriptional mechanisms. DNA methylation writers and readers regulate the activity-dependent genomic program required for memory consolidation. The most abundant DNA methylation reader, the Methyl CpG binding domain protein 2 (MeCP2), has been shown to regulate alternative splicing, but whether it establishes splicing events important for memory consolidation has not been investigated. In this study, we identified the alternative splicing profile of the mouse hippocampus in basal conditions and after a spatial learning experience, and investigated the requirement of MeCP2 for these processes. We observed that spatial learning triggers a wide-range of alternative splicing events in transcripts associated with structural and functional remodeling and that virus-mediated knockdown of MeCP2 impairs learning-dependent post-transcriptional responses of mature hippocampal neurons. Furthermore, we found that MeCP2 preferentially affected the splicing modalities intron retention and exon skipping and guided the alternative splicing of distinct set of genes in baseline conditions and after learning. Lastly, comparative analysis of the MeCP2-regulated transcriptome with the alternatively spliced mRNA pool, revealed that MeCP2 disruption alters the relative abundance of alternatively spliced isoforms without affecting the overall mRNA levels. Taken together, our findings reveal that adult hippocampal MeCP2 is required to finetune alternative splicing events in basal conditions, as well as in response to spatial learning. This study provides new insight into how MeCP2 regulates brain function, particularly cognitive abilities, and sheds light onto the pathophysiological mechanisms of Rett syndrome, that is characterized by intellectual disability and caused by mutations in the Mecp2 gene.