Primary Cultures Of Hippocampal Neurons Research Articles

A Method for Bioluminescence-Based RNA Monitoring Using Split-Luciferase Reconstitution Techniques.

A bioluminescence-based RNA monitoring method in living cells was developed using a split NanoLuc (NLuc) reconstitution technique. For specific recognition of a target RNA sequence, a mutant PUM-HD (mPUM) was used. The method was applied to β-actin mRNA in various cells, including primary cultures of rat hippocampal neurons. It allowed for continuous observation of intracellular localization distribution of the target mRNA in living cells, providing insights into various biological phenomena involving intracellular RNA localization and dynamics.

Perovskite Quantum Dot-Based Photovoltaic Biointerface for Photostimulation of Neurons.

A promising avenue for vision restoration against retinal degeneration is the use of semiconductor-based photovoltaic biointerfaces to substitute natural photoreceptors. Instead of silicon, perovskite has emerged as an exciting material for solar energy harvesting, and its nanocrystalline forms generally offer better stability than their bulk counterparts in addition to the distinct synthesis and fabrication steps. Herein, we synthesize tetramethylammonium lead iodide (TMAPbI3) perovskite quantum dots (QDs) as a novel photoactive material for photovoltaic biointerfaces. While the TMAPbI3 quantum dots and electrolyte interface induces Faradaic photocurrent under light illumination, the heterojunction with P3HT converts the charge-transfer process to a safe capacitive photocurrent with an improved ionic responsivity of 17.4 mA/W. The integration of the 18-nm quantum dot thickness shows good biocompatibility with primary cultures of hippocampal neurons and the photoresponse of the biointerface triggered photostimulation of the neurons. The rise of perovskite materials can stimulate novel forms of photovoltaic retina implants.

Effects of extracellular metabolic acidosis and out-of-equilibrium CO2/HCO3 - solutions on intracellular pH in cultured rat hippocampal neurons.

Metabolic acidosis (MAc)-an extracellular pH (pHo) decrease caused by a [HCO3 -]o decrease at constant [CO2]o-usually causes intracellular pH (pHi) to fall. Here we determine the extent to which the pHi decrease depends on the pHo decrease vs the concomitant [HCO3 -]o decrease. We use rapid-mixing to generate out-of-equilibrium CO2/HCO3 - solutions in which we stabilize [CO2]o and [HCO3 -]o while decreasing pHo (pure acidosis, pAc), or stabilize [CO2]o and pHo while decreasing [HCO3 -]o (pure metabolic/down, pMet↓). Using the fluorescent dye 2',7'-bis-2-carboxyethyl)-5(and-6)carboxyfluorescein (BCECF) to monitor pHi in rat hippocampal neurons in primary culture, we find that-in naïve neurons-the pHi decrease caused by MAc is virtually the sum of those caused by pAc (∼70%) + pMet↓ (∼30%). However, if we impose a first challenge (MAc1, pAc1, or pMet↓1), allow the neurons to recover, and then impose a second challenge (MAc2, pAc2, or pMet↓2), we find that pAc/pMet↓ additivity breaks down. In a twin-challenge protocol in which challenge #2 is MAc, the pHo and [HCO3 -]o decreases during challenge #1 must be coincident in order to mimic the effects of MAc1 on MAc2. Conversely, if challenge #1 is MAc, then the pHo and [HCO3 -]o decreases during challenge #2 must be coincident in order for MAc1 to produce its physiological effects during the challenge #2 period. We conclude that the history of challenge #1 (MAc1, pAc1, or pMet↓1)-presumably as detected by one or more acid-base sensors-has a major impact on the pHi response during challenge #2 (MAc2, pAc2, or pMet↓2).

A Nanobody-Based Proximity Ligation Assay Detects Constitutive and Stimulus-Regulated Native Arc/Arg3.1 Oligomers in Hippocampal Neuronal Dendrites.

Activity-regulated cytoskeleton-associatedprotein (Arc), the product of an immediate early gene, plays critical roles in synaptic plasticity and memory. Evidence suggests that Arc function is determined by its oligomeric state; however, methods for localization of native Arc oligomers are lacking. Here, we developed a nanobody-based proximity ligation assay (PLA) for detection, localization, and quantification of Arc-Arc complexes in primary rat hippocampal neuronal cultures. We used nanobodies with single, structurally defined epitopes in the bilobar Arc capsid domain. Nanobody H11 binds inside the N-lobe ligand pocket, while nanobody C11 binds to the C-lobe surface. For each nanobody, ALFA- and FLAG-epitope tags created a platform for antibody binding and PLA. Surprisingly, PLA puncta in neuronal dendrites revealed widespread constitutive Arc-Arc complexes. Treatment of cultures with tetrodotoxin or cycloheximide had no effect, suggesting stable complexes that are independent of recent neuronal activity and protein synthesis. To assess detection of oligomers, cultures were exposed to a cell-penetrating peptide inhibitor of the Arc oligomerization motif (OligoOFF). Arc-Arc complexes detected by H11 PLA were inhibited by OligoOff but not by control peptide. Notably, Arc complexes detected by C11 were unaffected by OligoOFF. Furthermore, we evaluated Arc complex formation after chemical stimuli that increase Arc synthesis. Brain-derived neurotrophic factor increased Arc-Arc signal detected by C11, but not H11. Conversely, dihydroxyphenylglycine (DHPG) treatment selectively enhanced H11 PLA signals. In sum, nanobody-based PLA reveals constitutive and stimulus-regulated Arc oligomers in hippocampal neuronal dendrites. A model is proposed based on detection of Arc dimer by C11 and higher-order oligomer by H11 nanobody.

Elastic Properties of the Cell Surface and Metabolic Profile of an Embryonic Primary Mixed Culture of Hippocampal Neurons under Conditions of P2X3 Receptor Blockade

Elastic Properties of the Cell Surface and Metabolic Profile of an Embryonic Primary Mixed Culture of Hippocampal Neurons under Conditions of P2X3 Receptor Blockade

A biallelic mutation in CACNA2D2 associated with developmental and epileptic encephalopathy affects calcium channel-dependent as well as synaptic functions of α2δ-2.

α2δ proteins serve as auxiliary subunits of voltage-gated calcium channels and regulate channel membrane expression and current properties. Besides their channel function, α2δ proteins regulate synapse formation, differentiation, and synaptic wiring. Considering these important functions, it is not surprising that CACNA2D1-4, the genes encoding for α2δ-1 to -4 isoforms, have been implicated in neurological, neurodevelopmental, and neuropsychiatric disorders. Mutations in CACNA2D2 have been associated with developmental and epileptic encephalopathy (DEE) and cerebellar atrophy. In our present study, we performed a detailed functional characterization of the p.R593P mutation in α2δ-2, a homozygous mutation previously identified in two siblings with DEE. Importantly, we analyzed both calcium channel-dependent as well as synaptic functions of α2δ-2. Our data show that the corresponding p.R596P mutation in mouse α2δ-2 drastically decreases membrane expression and synaptic targeting of α2δ-2. This defect correlates with altered biophysical properties of postsynaptic CaV1.3 channel but has no effect on presynaptic CaV2.1 channels upon heterologous expression in tsA201 cells. However, homologous expression of α2δ-2_R596P in primary cultures of hippocampal neurons affects the ability of α2δ-2 to induce a statistically significant increase in the presynaptic abundance of endogenous CaV2.1 channels and presynaptic calcium transients. Moreover, our data demonstrate that in addition to lowering membrane expression, the p.R596P mutation reduces the trans-synaptic recruitment of GABAA receptors and presynaptic synapsin clustering in glutamatergic synapses. Lastly, the α2δ-2_R596P reduces the amplitudes of glutamatergic miniature postsynaptic currents in transduced hippocampal neurons. Taken together, our data strongly link the human biallelic p.R593P mutation to the underlying severe neurodevelopmental disorder and highlight the importance of studying α2δ mutations not only in the context of channelopathies but also synaptopathies.

Delayed recruitment of activity-dependent bulk endocytosis in Fmr1 knockout neurons.

The presynapse performs an essential role in brain communication via the activity-dependent release of neurotransmitters. However, the sequence of events through which a presynapse acquires functionality is relatively poorly understood, which is surprising, since mutations in genes essential for its operation are heavily implicated in neurodevelopmental disorders. We addressed this gap in knowledge by determining the developmental trajectory of synaptic vesicle (SV) recycling pathways in primary cultures of rat hippocampal neurons. Exploiting a series of optical and morphological assays, we revealed that the majority of nerve terminals displayed activity-dependent calcium influx from 3 days invitro (DIV), immediately followed by functional evoked exocytosis and endocytosis, although the number of responsive nerve terminals continued to increase until the second week invitro. However, the most intriguing discovery was that activity-dependent bulk endocytosis (ADBE) was only observed from DIV 14 onwards. Importantly, optimal ADBE recruitment was delayed until DIV 21 in Fmr1 knockout neurons, which model Fragile X Syndrome (FXS). This implicates the delayed recruitment of ADBE as a potential contributing factor in the development of circuit dysfunction in FXS, and potentially other neurodevelopmental disorders.

Cytosolic Escape of Mitochondrial DNA Triggers cGAS-STING Pathway-Dependent Neuronal PANoptosis in Response to Intermittent Hypoxia.

Intermittent hypoxia (IH) is the predominant pathophysiological disturbance in obstructive sleep apnea (OSA), characterized by neuronal cell death and neurocognitive impairment. We focus on the accumulated mitochondrial DNA (mtDNA) in the cytosol, which acts as a damage-associated molecular pattern (DAMP) and activates the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, a known trigger for immune responses and neuronal death in degenerative diseases. However, the specific role and mechanism of the mtDNA-cGAS-STING axis in IH-induced neural damage remain largely unexplored. Here, we investigated the involvement of PANoptosis, a novel type of programmed cell death linked to cytosolic mtDNA accumulation and the cGAS-STING pathway activation, in neuronal cell death induced by IH. Our study found that PANoptosis occurred in primary cultures of hippocampal neurons and HT22 cell lines exposed to IH. In addition, we discovered that during IH, mtDNA released into the cytoplasm via the mitochondrial permeability transition pore (mPTP) activates the cGAS-STING pathway, exacerbating PANoptosis-associated neuronal death. Pharmacologically inhibiting mPTP opening or depleting mtDNA significantly reduced cGAS-STING pathway activation and PANoptosis in HT22 cells under IH. Moreover, our findings indicated that the cGAS-STING pathway primarily promotes PANoptosis by modulating endoplasmic reticulum (ER) stress. Inhibiting or silencing the cGAS-STING pathway substantially reduced ER stress-mediated neuronal death and PANoptosis, while lentivirus-mediated STING overexpression exacerbated these effects. In summary, our study elucidates that cytosolic escape of mtDNA triggers cGAS-STING pathway-dependent neuronal PANoptosis in response to IH, mainly through regulating ER stress. The discovery of the novel mechanism provides theoretical support for the prevention and treatment of neuronal damage and cognitive impairment in patients with OSA.

Mitochondrial respiration of a primary mixed culture of neurons from hippocampus at various stages of differentiation

BACKGROUND: Primary mixed culture of neurons is often used to evaluate the molecular mechanisms underlying neurodegenerative disorders. However, isolating cells from the brain is accompanied by profound changes in cell morphology, behavior, and metabolic rate due to enzymatic disintegration and microenvironmental changes in cells. In this regard, the mitochondrial basal respiration of neurons should be considered as an indicator of the formation of functional activity of the cell.
 AIM: To evaluate mitochondrial respiration in a primary mixed culture of hippocampal neurons at various stages of differentiation.
 METHODS: This study included mice of line CD-1 and was conducted in two series: the first studied the primary mixed embryonic culture on the 18th day of gestation (E18) and the second used the postnatal culture on the 2nd day after birth (P2). A Seahorse XF HS Mini (Agilent, USA) was used to measure the functional parameters of cell metabolism. The oxygen consumption rate was calculated based on the results of the mitochondrial respiration profile of the primary mixed culture built during its differentiation.
 RESULTS: On the 5th day of neuronal differentiation in the culture, maximum mitochondrial respiration was established both in the hippocampal culture obtained from embryos on the 18th day of gestation and in the culture taken from mice on the 2nd day after birth. As cells differentiated in culture from days 2 to 11, the substrate oxidation rate increased almost twofold in the culture of hippocampal neurons obtained from embryos, which increased the metabolic potential.
 CONCLUSION: The study results prove that the hippocampus can be used to study the role of mitochondria in neurogenesis.

The role of glia in the dysregulation of neuronal spinogenesis in Ube3a-dependent ASD

Overexpression of the Ube3a gene and the resulting increase in Ube3a protein are linked to autism spectrum disorder (ASD). However, the cellular and molecular processes underlying Ube3a-dependent ASD remain unclear. Using both male and female mice, we find that neurons in the somatosensory cortex of the Ube3a 2× Tg ASD mouse model display reduced dendritic spine density and increased immature filopodia density. Importantly, the increased gene dosage of Ube3a in astrocytes alone is sufficient to confer alterations in neurons as immature dendritic protrusions, as observed in primary hippocampal neuron cultures. We show that Ube3a overexpression in astrocytes leads to a loss of astrocyte-derived spinogenic protein, thrombospondin-2 (TSP2), due to a suppression of TSP2 gene transcription. By neonatal intraventricular injection of astrocyte-specific virus, we demonstrate that Ube3a overexpression in astrocytes in vivo results in a reduction in dendritic spine maturation in prelimbic cortical neurons, accompanied with autistic-like behaviors in mice. These findings reveal an astrocytic dominance in initiating ASD pathobiology at the neuronal and behavior levels. Significance statementIncreased gene dosage of Ube3a is tied to autism spectrum disorders (ASDs), yet cellular and molecular alterations underlying autistic phenotypes remain unclear. We show that Ube3a overexpression leads to impaired dendritic spine maturation, resulting in reduced spine density and increased filopodia density. We find that dysregulation of spine development is not neuron autonomous, rather, it is mediated by an astrocytic mechanism. Increased gene dosage of Ube3a in astrocytes leads to reduced production of the spinogenic glycoprotein thrombospondin-2 (TSP2), leading to abnormalities in spines. Astrocyte-specific Ube3a overexpression in the brain in vivo confers dysregulated spine maturation concomitant with autistic-like behaviors in mice. These findings indicate the importance of astrocytes in aberrant neurodevelopment and brain function in Ube3a-depdendent ASD.

Human mutations in SLITRK3 implicated in GABAergic synapse development in mice.

This study reports on biallelic homozygous and monoallelic de novo variants in SLITRK3 in three unrelated families presenting with epileptic encephalopathy associated with a broad neurological involvement characterized by microcephaly, intellectual disability, seizures, and global developmental delay. SLITRK3 encodes for a transmembrane protein that is involved in controlling neurite outgrowth and inhibitory synapse development and that has an important role in brain function and neurological diseases. Using primary cultures of hippocampal neurons carrying patients' SLITRK3 variants and in combination with electrophysiology, we demonstrate that recessive variants are loss-of-function alleles. Immunostaining experiments in HEK-293 cells showed that human variants C566R and E606X change SLITRK3 protein expression patterns on the cell surface, resulting in highly accumulating defective proteins in the Golgi apparatus. By analyzing the development and phenotype of SLITRK3 KO (SLITRK3-/-) mice, the study shows evidence of enhanced susceptibility to pentylenetetrazole-induced seizure with the appearance of spontaneous epileptiform EEG as well as developmental deficits such as higher motor activities and reduced parvalbumin interneurons. Taken together, the results exhibit impaired development of the peripheral and central nervous system and support a conserved role of this transmembrane protein in neurological function. The study delineates an emerging spectrum of human core synaptopathies caused by variants in genes that encode SLITRK proteins and essential regulatory components of the synaptic machinery. The hallmark of these disorders is impaired postsynaptic neurotransmission at nerve terminals; an impaired neurotransmission resulting in a wide array of (often overlapping) clinical features, including neurodevelopmental impairment, weakness, seizures, and abnormal movements. The genetic synaptopathy caused by SLITRK3 mutations highlights the key roles of this gene in human brain development and function.

Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics

BackgroundRasal1 is a Ras GTPase-activating protein which contains C2 domains necessary for dynamic membrane association following intracellular calcium elevation. Membrane-bound Rasal1 inactivates Ras signaling through its RasGAP activity, and through such mechanisms has been implicated in regulating various cellular functions in the context of tumors. Although highly expressed in the brain, the contribution of Rasal1 to neuronal development and function has yet to be explored.ResultsWe examined the contributions of Rasal1 to neuronal development in primary culture of hippocampal neurons through modulation of Rasal1 expression using molecular tools. Fixed and live cell imaging demonstrate diffuse expression of Rasal1 throughout the cell soma, dendrites and axon which localizes to the neuronal plasma membrane in response to intracellular calcium fluctuation. Pull-down and co-immunoprecipitation demonstrate direct interaction of Rasal1 with PKC, tubulin, and CaMKII. Consequently, Rasal1 is found to stabilize microtubules, through post-translational modification of tubulin, and accordingly inhibit dendritic outgrowth and branching. Through imaging, molecular, and electrophysiological techniques Rasal1 is shown to promote NMDA-mediated synaptic activity and CaMKII phosphorylation.ConclusionsRasal1 functions in two separate roles in neuronal development; calcium regulated neurite outgrowth and the promotion of NMDA receptor-mediated postsynaptic events which may be mediated both by interaction with direct binding partners or calcium-dependent regulation of down-stream pathways. Importantly, the outlined molecular mechanisms of Rasal1 may contribute notably to normal neuronal development and synapse formation.

The PTB and PRR domains of numb regulate neurite outgrowth by influencing voltage-gated calcium channel expression and kinetics

Numb is an evolutionarily conserved protein that regulates the differentiation of neuronal progenitor cells through unknown mechanisms. Numb has four alternative splice variants with different lengths of phosphotyrosine-binding (PTB) and proline-rich regions (PRR) domains. In this study, we demonstrated that Numb expression was increased in the primary cultures of rat cortical and hippocampal neurons over time in vitro, and Numb antisense inhibited neurite outgrowth. We verified that cells overexpressing short PTB (SPTB) or long PTB (LPTB) domains exhibited differentiation or proliferation, respectively. SPTB-mediated differentiation was related to the PRR domains, as cells expressing SPTB/LPRR had longer dendrites and more branched dendrites than cells expressing SPTB/SPRR. The differentiation of both cell types was completely blocked by the Ca2+ chelator. Western blot analysis revealed the increased total protein expression of voltage-gated calcium channel (VGCC) subunit α1C and α1D in cells expressing SPTB and LPTB Numb. The increased expression of the VGCC β3 subunit was only observed in cells expressing SPTB Numb. Immunocytochemistry further showed that SPTB-mediated cell differentiation was associated with increased membrane expression of VGCC subunits α1C, α1D and β3, which corresponded to the higher Ca2+ current (ICa) densities. Furthermore, we found that VGCC of cells transfected with SPTB/SPRR or SPTB/LPRR Numb isoforms exhibit steady-state inactivation (SSI) in both differentiated and undifferentiated phenotypes. A similar SSI of VGCC was observed in the differentiated cells transfected with SPTB/SPRR or SPTB/LPRR Numb isoforms, whereas a left shift SSI of VGCC in cells expressing SPTB/LPRR was detected in the undifferentiated cells. Collectively, these data indicate that SPTB domain is essential for neurite outgrowth involving in membrane expression of VGCC subunits, and LPRR plays a role in neuronal branching and the regulation of VGCC inactivation kinetics.

Changes in structural plasticity of hippocampal neurons in an animal model of multiple sclerosis.

Structural plasticity is critical for the functional diversity of neurons in the brain. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used model for multiple sclerosis (MS), successfully mimicking its key pathological features (inflammation, demyelination, axonal loss, and gliosis) and clinical symptoms (motor and non-motor dysfunctions). Recent studies have demonstrated the importance of synaptic plasticity in EAE pathogenesis. In the present study, we investigated the features of behavioral alteration and hippocampal structural plasticity in EAE-affected mice in the early phase (11 days post-immunization, DPI) and chronic phase (28 DPI). EAE-affected mice exhibited hippocampus-related behavioral dysfunction in the open field test during both early and chronic phases. Dendritic complexity was largely affected in the cornu ammonis 1 (CA1) and CA3 apical and dentate gyrus (DG) subregions of the hippocampus during the chronic phase, while this effect was only noted in the CA1 apical subregion in the early phase. Moreover, dendritic spine density was reduced in the hippocampal CA1 and CA3 apical/basal and DG subregions in the early phase of EAE, but only reduced in the DG subregion during the chronic phase. Furthermore, mRNA levels of proinflammatory cytokines ( Il1β, Tnfα, and Ifnγ) and glial cell markers ( Gfap and Cd68) were significantly increased, whereas the expression of activity-regulated cytoskeleton-associated protein (ARC) was reduced during the chronic phase. Similarly, exposure to the aforementioned cytokines in primary cultures of hippocampal neurons reduced dendritic complexity and ARC expression. Primary cultures of hippocampal neurons also showed significantly reduced extracellular signal-regulated kinase (ERK) phosphorylation upon treatment with proinflammatory cytokines. Collectively, these results suggest that autoimmune neuroinflammation alters structural plasticity in the hippocampus, possibly through the ERK-ARC pathway, indicating that this alteration may be associated with hippocampal dysfunctions in EAE.

Mitochondrial calcium function is altered by lithium in hippocampal cell culture and triple transgenic for Alzheimer’s disease

AbstractBackgroundCalcium (Ca2+) regulate many functions in neuronal and glial cells. The Mitochondrial Calcium Uniporter (Mcu) complex is thought to be the primary pathway for Ca2+ entry into mitochondria. The Mcu protein has an important role in regulating mitochondrial Ca2+ homeostasis. Dysregulation of mitochondrial Ca2+ homeostasis has been implicated in various pathological conditions, such as Alzheimer disease. AD pathology is characterized by the progressive accumulation of amyloid β‐peptide and neurofibrillary tangles. In AD patients a significant increase in an oxidative damage and decrease in energy metabolism resulting in a mitochondrial calcium disbalance. Lithium has been used for decades to treat neurodegenerative diseases, as it has an action on several intracellular organelles, including mitochondria. The lithium regulates calcium channels, decreases oxidative stress and increases autophagic processes. Therefore, we aim to analyze whether chronic treatment with lithium in primary cultures of hippocampal neurons and in the hippocampus of triple transgenic animals for Alzheimer’s disease (3xtgAD).MethodThe cultures were treated for 7 days (dose of 0.02; 0.2 and 2mM) and 3xtgAD for 8 months (doses of 1mM and 2mM). The hippocampus were analysed by proteomic and transcriptomics.ResultOur results showed that lithium increases Mcu and Micu 3 proteins, showing that lithium has an effect on the modulation of mitochondrial calcium channels that should be explored.ConclusionMitochondrial calcium is an important mechanism for understanding neurodegenerative diseases and lithium can reverse Alzheimer’s disease‐related dysfunction.

Effects of lithium on phosphatidyinositol‐3‐kinase signaling pathways in triple transgenicanimals of Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease (AD), a severe, progressive and multifactorial disease. Among the altered biological pathways, we have the phosphatidyl inositol 3 kinase (PI3K), an important cell signaling mediator that loses its function as a result of changes in the Tau protein and Aβ toxicity. Our aim is to determine the ontological pathways related to PI3K in 3xTg‐AD animals chronically treated with lithium at therapeutic and subtherapeutic doses. Also determine the unique, statistically significant proteins of the biological pathways in the ontological pathway altered in hippocampal tissue.MethodThe animals' hippocampus (WT and 3xTg‐AD) were divided into three subgroups: Li0 (standard chow); Li1 (ration containing 1.0 g of lithium carbonate/Kg); and Li2 (ration containing 2.0 g of lithium carbonate/Kg). Primary cultures of hippocampal neurons were treated with doses of 0.02; 0. 2 and 2mM. The identified protein pathways will be compared by Gene Ontology, using ClueGO, a Cytoscape software plug‐in. The analyzes for comparison with neurons and subsequent tissue identification will be performed by ‘Benjamini & Hochberg False Discovery Rate’ (p≤0.05 setting) for test correction for ‘biological process’. Ethical committee (1293/09).ResultOur results on PI3K showed enrichment of pathways: response to hormone, cell migration, cellular response to endogenous stimulus, positive regulation of response to stimulus and regulation of catalytic activity.ConclusionInitial results are promising for the pharmacological understanding of lithium as a neuroprotector for Alzheimer’s disease.

Effects of lithium on phosphatidylinositol‐3‐kinase signaling pathways in triple transgenic animals for Alzheimer' disease

AbstractBackgroundAlzheimer’s disease (AD), a severe, progressive and multifactorial disease. Among the altered biological pathways, we have the phosphatidyl inositol 3 kinase (PI3K), an important cell signaling mediator that loses its function as a result of changes in the Tau protein and Aβ toxicity. Our aim is to determine the ontological pathways related to PI3K in 3xTg‐AD animals chronically treated with lithium at therapeutic and subtherapeutic doses. Also determine the unique, statistically significant proteins of the biological pathways in the ontological pathway altered in hippocampal tissue.MethodThe animals' hippocampus (WT and 3xTg‐AD) were divided into three subgroups: Li0 (standard chow); Li1 (ration containing 1.0 g of lithium carbonate/Kg); and Li2 (ration containing 2.0 g of lithium carbonate/Kg). Primary cultures of hippocampal neurons were treated with doses of 0.02; 0. 2 and 2mM. The identified protein pathways will be compared by Gene Ontology, using ClueGO, a Cytoscape software plug‐in. The analyzes for comparison with neurons and subsequent tissue identification will be performed by ‘Benjamini & Hochberg False Discovery Rate’ (p≤0.05 setting) for test correction for ‘biological process’. Ethical committee (1293/09).ResultOur results on PI3K showed enrichment of pathways: response to hormone, cell migration, cellular response to endogenous stimulus, positive regulation of response to stimulus and regulation of catalytic activity.ConclusionInitial results are promising for the pharmacological understanding of lithium as a neuroprotector for Alzheimer’s disease.

Cellular Iron Deficiency Disrupts Thyroid Hormone Regulated Gene Expression in Developing Hippocampal Neurons

BackgroundDeveloping neurons have high thyroid hormone and iron requirements to support their metabolically demanding growth. Early-life iron and thyroid-hormone deficiencies are prevalent and often coexist, and each independently increases risk of permanently impaired neurobehavioral function in children. Early-life dietary iron deficiency reduces thyroid-hormone concentrations and impairs thyroid hormone-responsive gene expression in the neonatal rat brain, but it is unclear whether the effect is cell-intrinsic. ObjectivesThis study determined whether neuronal-specific iron deficiency alters thyroid hormone-regulated gene expression in developing neurons. MethodsIron deficiency was induced in primary mouse embryonic hippocampal neuron cultures with the iron chelator deferoxamine (DFO) beginning at 3 d in vitro (DIV). At 11DIV and 18DIV, thyroid hormone-regulated gene messenger ribonucleic acid (mRNA)concentrations indexing thyroid hormone homeostasis (Hairless, mu-crystallin, Type II deiodinase, solute carrier family member 1c1, and solute carrier family member 16a2) and neurodevelopment (neurogranin, Parvalbumin, and Krüppel-like factor 9) were quantified. To assess the effect of iron repletion, DFO was removed at 14DIV from a subset of DFO-treated cultures, and gene expression and adenosine 5'-triphosphate (ATP) concentrations were quantified at 21DIV. ResultsAt 11DIV and 18DIV, neuronal iron deficiency decreased neurogranin, Parvalbumin, and mu-crystallin, and by 18DIV, solute carrier family member 16a2, solute carrier family member 1c1, Type II deiodinase, and Hairless were increased, suggesting cellular sensing of a functionally abnormal thyroid hormone state. Dimensionality reduction with Principal component analysis reveals that thyroid hormone homeostatic genes strongly correlate with and predict iron status. Iron repletion from 14–21DIV did not restore ATP concentration, and Principal component analysis suggests that, after iron repletion, cultures maintain a gene expression signature indicative of previous iron deficiency. ConclusionsThese novel findings suggest there is an intracellular mechanism coordinating cellular iron/thyroid hormone activities. We speculate this is a part of the homeostatic response to acutely match neuronal energy production and growth signaling. However, the adaptation to iron deficiency may cause permanent deficits in thyroid hormone-dependent neurodevelopmental processes even after recovery from iron deficiency.

Clinical and electrophysiological features of SCN8A variants causing episodic or chronic ataxia

Variants in SCN8A are associated with a spectrum of epilepsies and neurodevelopmental disorders. Ataxia as a predominant symptom of SCN8A variation has not been well studied. We set out to investigate disease mechanisms and genotype-phenotype correlations of SCN8A-related ataxia. We collected genetic and electro-clinical data of ten individuals from nine unrelated families carrying novel SCN8A variants associated with chronic progressive or episodic ataxia. Electrophysiological characterizations of these variants were performed in ND7/23cells and cultured neurons. Variants associated with chronic progressive ataxia either decreased Na+ current densities and shifted activation curves towards more depolarized potentials (p.Asn995Asp, p.Lys1498Glu and p.Trp1266Cys) or resulted in a premature stop codon (p.Trp937Ter). Three variants (p.Arg847Gln and biallelic p.Arg191Trp/p.Asp1525Tyr) were associated with episodic ataxia causing loss-of-function by decreasing Na+ current densities or a hyperpolarizing shift of the inactivation curve. Two additional episodic ataxia-associated variants caused mixed gain- and loss-of function effects in ND7/23cells and were further examined in primary murine hippocampal neuronal cultures. Neuronal firing in excitatory neurons was increased by p.Arg1629His, but decreased by p.Glu1201Lys. Neuronal firing in inhibitory neurons was decreased for both variants. No functional effect was observed for p.Arg1913Trp. In four individuals, treatment with sodium channel blockers exacerbated symptoms. We identified episodic or chronic ataxia as predominant phenotypes caused by variants in SCN8A. Genotype-phenotype correlations revealed a more pronounced loss-of-function effect for variants causing chronic ataxia. Sodium channel blockers should be avoided under these conditions. BMBF, DFG, the Italian Ministry of Health, University of Tuebingen.

K+/Cl- cotransporter 2 (KCC2) and Na+/ cotransporter 1 (NBCe1) interaction modulates profile of KCC2 phosphorylation.

K+/Cl- cotransporter 2 (KCC2) is a major Cl- extruder in mature neurons and is responsible for the establishment of low intracellular [Cl-], necessary for fast hyperpolarizing GABAA-receptor mediated synaptic inhibition. Electrogenic sodium bicarbonate cotransporter 1 (NBCe1) is a pH regulatory protein expressed in neurons and glial cells. An interactome study identified NBCe1 as a possible interaction partner of KCC2. In this study, we investigated the putative effect of KCC2/NBCe1 interaction in baseline and the stimulus-induced phosphorylation pattern and function of KCC2. Primary mouse hippocampal neuronal cultures from wildtype (WT) and Nbce1-deficient mice, as well as HEK-293 cells stably transfected with KCC2WT, were used. The results show that KCC2 and NBCe1 are interaction partners in the mouse brain. In HEKKCC2 cells, pharmacological inhibition of NBCs with S0859 prevented staurosporine- and 4-aminopyridine (4AP)-induced KCC2 activation. In mature cultures of hippocampal neurons, however, S0859 completely inhibited postsynaptic GABAAR and, thus, could not be used as a tool to investigate the role of NBCs in GABA-dependent neuronal networks. In Nbce1-deficient immature hippocampal neurons, baseline phosphorylation of KCC2 at S940 was downregulated, compared to WT, and exposure to staurosporine failed to reduce pKCC2 S940 and T1007. In Nbce1-deficient mature neurons, baseline levels of pKCC2 S940 and T1007 were upregulated compared to WT, whereas after 4AP treatment, pKCC2 S940 was downregulated, and pKCC2 T1007 was further upregulated. Functional experiments showed that the levels of GABAAR reversal potential, baseline intracellular [Cl-], Cl- extrusion, and baseline intracellular pH were similar between WT and Nbce1-deficient neurons. Altogether, our data provide a primary description of the properties of KCC2/NBCe1 protein-protein interaction and implicate modulation of stimulus-mediated phosphorylation of KCC2 by NBCe1/KCC2 interaction-a mechanism with putative pathophysiological relevance.