Mechanisms of synapse-to-nucleus calcium signalling in striatal neurons and impairments in Huntington's disease.

Huntington's disease (HD) is a monogenic disorder with autosomal dominant inheritance. In HD patients, neurons in the striatum and cortex degenerate, leading to motor, psychiatric and cognitive disorders. Dysregulated synaptic function and calcium handling are common in many neurodegenerative diseases, including HD. N-methyl-D-aspartate (NMDA) receptor function is enhanced in HD at extrasynaptic sites, altering the balance of calcium-dependent neuronal survival versus death signalling pathways. Endoplasmic reticulum (ER) calcium handling is also abnormal in HD. The ER, which is continuous with the nuclear envelope, is purportedly involved in nuclear calcium signalling; based on this, we hypothesised that nuclear calcium signalling is altered in HD. We explored this hypothesis with calcium imaging techniques, including simultaneous epifluorescent imaging of cytosolic and nuclear calcium using jRCaMP1b and GCaMP3 sensors, respectively, in striatal spiny projection neurons in cortical-striatal co-cultures from the YAC128 mouse model of HD. Our data show contributions from a variety of calcium channels to nuclear calcium signalling. NMDA receptors (NMDARs) play an essential role in initiating action potential-dependent calcium signalling to the nucleus, and ryanodine receptors (RyR) contribute to both cytosolic and nuclear calcium signals. Unlike previous reports in glutamatergic hippocampal and cortical neurons, we found that in GABAergic striatal neurons, L-type voltage-gated calcium channels (CaV) contribute to cytosolic, but not nuclear calcium signalling. Calcium imaging also suggests impairments in nuclear calcium signalling in HD striatal neurons, where spontaneous action potential-dependent calcium transients in the nucleus were smaller in YAC128 striatal neurons compared to those of wild-type (WT). Our results elucidate mechanisms involved in action potential-dependent nuclear calcium signalling in GABAergic striatal neurons, and have revealed a clear deficit in this signalling in HD.

Phase-amplitude coupling detection and analysis of human 2-dimensional neural cultures in multi-well microelectrode array in vitro.

BACKGROUND: Human induced pluripotent stem cell (hiPSC)- derived neurons offer the possibility of studying human-specific neuronal behaviors in physiologic and pathologic states in vitro. It is unclear whether cultured neurons can achieve the fundamental network behaviors required to process information in the brain. Investigating neuronal oscillations and their interactions, as occurs in cross-frequency coupling (CFC), addresses this question. NEW METHODS: We examined whether networks of two-dimensional (2D) cultured hiPSC-derived cortical neurons grown with hiPSC-derived astrocytes on microelectrode array plates recapitulate the CFC that is present in vivo. We employed the modulation index method for detecting phase-amplitude coupling (PAC) and used offline spike sorting to analyze the contribution of single neuron spiking to network behavior. RESULTS: We found that PAC is present, the degree of PAC is specific to network structure, and it is modulated by external stimulation with bicuculline administration. Modulation of PAC is not driven by single neurons, but by network-level interactions. COMPARISON WITH EXISTING METHODS: PAC has been demonstrated in multiple regions of the human cortex as well as in organoids. This is the first report of analysis demonstrating the presence of coupling in 2D cultures. CONCLUSION: CFC in the form of PAC analysis explores communication and integration between groups of neurons and dynamical changes across networks. In vitro PAC analysis has the potential to elucidate the underlying mechanisms as well as capture the effects of chemical, electrical, or ultrasound stimulation; providing insight into modulation of neural networks to treat nervous system disorders in vivo.

Retigabine and gabapentin restore channel function and neuronal firing in a cellular model of an epilepsy-associated dominant-negative KCNQ5 variant.

KCNQ5 encodes the voltage-gated potassium channel KV7.5, a member of the KV7 channel family, which conducts the M-current. This current is a potent regulator of neuronal excitability by regulating membrane potential in the subthreshold range of action potentials and mediating the medium and slow afterhyperpolarization. Recently, we have identified five loss-of-function variants in KCNQ5 in patients with genetic generalized epilepsy. Using the most severe dominant-negative variant (R359C), we set out to investigate pharmacological therapeutic intervention by KV7 channel openers on channel function and neuronal firing. Retigabine and gabapentin increased R359C-derived M-current amplitudes in HEK cells expressing homomeric or heteromeric mutant KV7.5 channels. Retigabine was most effective in restoring K+ currents. Ten µM retigabine was sufficient to reach the level of WT currents without retigabine, whereas 100 µM of gabapentin showed less than half of this effect and application of 50 µM ZnCl2 only significantly increased M-current amplitude in heteromeric channels. Overexpression of KV7.5-WT potently inhibited neuronal firing by increasing the M-current, whereas R359C overexpression had the opposite effect and additionally decreased the medium afterhyperpolarization current. Both aforementioned drugs and Zn2+ reversed the effect of R359C expression by reducing firing to nearly normal levels at high current injections. Our study shows that a dominant-negative variant with a complete loss-of-function in KV7.5 leads to largely increased neuronal firing which may explain a neuronal hyperexcitability in patients. KV7 channel openers, such as retigabine or gabapentin, could be treatment options for patients currently displaying pharmacoresistant epilepsy and carrying loss-of-function variants in KCNQ5.

Redox Protein Thioredoxin Mediates Neurite Outgrowth in Primary Cultured Mouse Cerebral Cortical Neurons.

Thioredoxin system plays an important role in maintaining the cellular redox balance. Recent evidence suggests that thioredoxin (Trx) system may promote cell survival and neuroprotection. In this study, we explored the role of thioredoxin system in neuronal differentiation using a primary mouse cortical neuronal cell culture. First, Trx and Trx reductase (TrxR) protein levels were analyzed in cultured neurons from 1 to 32 days in vitro (DIV). The result showed that Trx and TrxR protein levels time-dependently increased in the neuron cell culture from 1 to 18 DIV. To establish the role of Trx in neuronal differentiation, Trx gene expression was knockdown in cultured neurons using Trx sgRNA CRISPR/Cas9 technology. Treatment with CRISPR/Cas9/Trx sgRNA decreased Trx protein levels and caused a reduction in dendritic outgrowth and branching of cultured neurons. Then, primary cortical neurons were treated with the Trx inhibitor PX12 to block Trx reducing activity. Treatment with PX12 also reduced dendritic outgrowth and branching. Furthermore, PX12 treatment reduced the ratio of phosphorylated cyclic AMP response element-binding protein (CREB)/total CREB protein levels. To investigate whether CREB phosphorylation is redox regulated, SH-SY5Y cells were treated with H2O2, which reduced phosphorylated CREB protein levels and increased CREB thiol oxidation. However, treatment with CB3, a Trx-mimetic tripeptide, rescued H2O2-decreased CREB phosphorylation. Our results suggest that Trx regulates neuronal differentiation and maturation of primary mouse cortical neurons by targeting CREB neurotrophic pathway. Trx may regulate CREB activation by maintaining the cellular redox balance.

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

BACKGROUND: Developing 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. OBJECTIVES: This study determined whether neuronal-specific iron deficiency alters thyroid hormone-regulated gene expression in developing neurons. METHODS: Iron 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. RESULTS: At 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. CONCLUSIONS: These 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.

Drosophila CASK regulates brain size and neuronal morphogenesis, providing a genetic model of postnatal microcephaly suitable for drug discovery.

BACKGROUND: CASK-related neurodevelopmental disorders are untreatable. Affected children show variable severity, with microcephaly, intellectual disability (ID), and short stature as common features. X-linked human CASK shows dosage sensitivity with haploinsufficiency in females. CASK protein has multiple domains, binding partners, and proposed functions at synapses and in the nucleus. Human and Drosophila CASK show high amino-acid-sequence similarity in all functional domains. Flies homozygous for a hypomorphic CASK mutation (∆18) have motor and cognitive deficits. A Drosophila genetic model of CASK-related disorders could have great scientific and translational value. METHODS: We assessed the effects of CASK loss of function on morphological phenotypes in Drosophila using established genetic, histological, and primary neuronal culture approaches. NeuronMetrics software was used to quantify neurite-arbor morphology. Standard nonparametric statistics methods were supplemented by linear mixed effects modeling in some cases. Microfluidic devices of varied dimensions were fabricated and numerous fluid-flow parameters were used to induce oscillatory stress fields on CNS tissue. Dissociation into viable neurons and neurite outgrowth in vitro were assessed. RESULTS: We demonstrated that ∆18 homozygous flies have small brains, small heads, and short bodies. When neurons from developing CASK-mutant CNS were cultured in vitro, they grew small neurite arbors with a distinctive, quantifiable "bushy" morphology that was significantly rescued by transgenic CASK+. As in humans, the bushy phenotype showed dosage-sensitive severity. To overcome the limitations of manual tissue trituration for neuronal culture, we optimized the design and operation of a microfluidic system for standardized, automated dissociation of CNS tissue into individual viable neurons. Neurons from CASK-mutant CNS dissociated in the microfluidic system recapitulate the bushy morphology. Moreover, for any given genotype, device-dissociated neurons grew larger arbors than did manually dissociated neurons. This automated dissociation method is also effective for rodent CNS. CONCLUSIONS: These biological and engineering advances set the stage for drug discovery using the Drosophila model of CASK-related disorders. The bushy phenotype provides a cell-based assay for compound screening. Nearly a dozen genes encoding CASK-binding proteins or transcriptional targets also have brain-development mutant phenotypes, including ID. Hence, drugs that improve CASK phenotypes might also benefit children with disorders due to mutant CASK partners.

Synapses do not facilitate prion-like transfer of alpha-synuclein: a quantitative study in reconstructed unidirectional neural networks.

Alpha-synuclein (aSyn) aggregation spreads between cells and underlies the progression of neuronal lesions in the brain of patients with synucleinopathies such as Parkinson's diseases. The mechanisms of cell-to-cell propagation of aggregates, which dictate how aggregation progresses at the network level, remain poorly understood. Notably, while prion and prion-like spreading is often simplistically envisioned as a "domino-like" spreading scenario where connected neurons sequentially propagate protein aggregation to each other, the reality is likely to be more nuanced. Here, we demonstrate that the spreading of preformed aSyn aggregates is a limited process that occurs through molecular sieving of large aSyn seeds. We further show that this process is not facilitated by synaptic connections. This was achieved through the development and characterization of a new microfluidic platform that allows reconstruction of binary fully oriented neuronal networks in vitro with no unwanted backward connections, and through the careful quantification of fluorescent aSyn aggregates spreading between neurons. While this allowed us for the first time to extract quantitative data of protein seeds dissemination along neural pathways, our data suggest that prion-like dissemination of proteinopathic seeding aggregates occurs very progressively and leads to highly compartmentalized pattern of protein seeding in neural networks.

Treatment of Status Epilepticus after Traumatic Brain Injury Using an Antiseizure Drug Combined with a Tissue Recovery Enhancer Revealed by Systems Biology.

We tested a hypothesis that in silico-discovered compounds targeting traumatic brain injury (TBI)-induced transcriptomics dysregulations will mitigate TBI-induced molecular pathology and augment the effect of co-administered antiseizure treatment, thereby alleviating functional impairment. In silico bioinformatic analysis revealed five compounds substantially affecting TBI-induced transcriptomics regulation, including calpain inhibitor, chlorpromazine, geldanamycin, tranylcypromine, and trichostatin A (TSA). In vitro exposure of neuronal-BV2-microglial co-cultures to compounds revealed that TSA had the best overall neuroprotective, antioxidative, and anti-inflammatory effects. In vivo assessment in a rat TBI model revealed that TSA as a monotherapy (1 mg/kg/d) or in combination with the antiseizure drug levetiracetam (LEV 150 mg/kg/d) mildly mitigated the increase in plasma levels of the neurofilament subunit pNF-H and cortical lesion area. The percentage of rats with seizures during 0-72 h post-injury was reduced in the following order: TBI-vehicle 80%, TBI-TSA (1 mg/kg) 86%, TBI-LEV (54 mg/kg) 50%, TBI-LEV (150 mg/kg) 40% (p < 0.05 vs. TBI-vehicle), and TBI-LEV (150 mg/kg) combined with TSA (1 mg/kg) 30% (p < 0.05). Cumulative seizure duration was reduced in the following order: TBI-vehicle 727 ± 688 s, TBI-TSA 898 ± 937 s, TBI-LEV (54 mg/kg) 358 ± 715 s, TBI-LEV (150 mg/kg) 42 ± 64 (p < 0.05 vs. TBI-vehicle), and TBI-LEV (150 mg/kg) combined with TSA (1 mg/kg) 109 ± 282 s (p < 0.05). This first preclinical intervention study on post-TBI acute seizures shows that a combination therapy with the tissue recovery enhancer TSA and LEV was safe but exhibited no clear benefit over LEV monotherapy on antiseizure efficacy. A longer follow-up is needed to confirm the possible beneficial effects of LEV monotherapy and combination therapy with TSA on chronic post-TBI structural and functional outcomes, including epileptogenesis.

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

Background: Developing neurons have high thyroid hormone and iron requirements to support their metabolism and growth. Early-life iron and thyroid hormone deficiencies are prevalent, often coexist, and increase the risk of permanently impaired neurobehavioral function in children. Early-life dietary iron deficiency reduces thyroid hormone levels and impairs thyroid hormone-responsive gene expression in the neonatal rat brain. Objective: This study determined whether neuronal-specific iron deficiency alters thyroid hormone-regulated gene expression in developing neurons. Methods: Iron deficiency was induced in primary mouse embryonic hippocampal neuron cultures with the iron chelator deferoxamine (DFO) beginning at 3 days in vitro (DIV). At 11DIV and 18DIV, mRNA levels for thyroid hormone-regulated genes indexing thyroid hormone homeostasis (Hr, Crym, Dio2, Slco1c1, Slc16a2) and neurodevelopment (Nrgn, Pvalb, Klf9) 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 ATP levels were quantified at 21DIV. Results: At 11DIV and 18DIV, neuronal iron deficiency decreased Nrgn, Pvalb, and Crym, and by 18DIV, Slc16a2, Slco1c1, Dio2, and Hr were increased; collectively suggesting cellular sensing of a functionally abnormal thyroid hormone state. Dimensionality reduction with Principal Component Analysis (PCA) reveals that thyroid hormone homeostatic genes strongly correlate with and predict iron status (Tfr1 mRNA). Iron repletion from 14-21DIV restored neurodevelopmental genes, but not all thyroid hormone homeostatic genes, and ATP concentrations remained significantly altered. PCA clustering suggests that cultures replete with iron maintain a gene expression signature indicative of previous iron deficiency. Conclusions: These novel findings suggest there is an intracellular mechanism coordinating cellular iron/thyroid hormone activities. We speculate this is a part of homeostatic response to match neuronal energy production and growth signaling for these important metabolic regulators. However, iron deficiency may cause permanent deficits in thyroid hormone-dependent neurodevelopmental processes even after recovery from iron deficiency.

Live-Imaging Detection of Multivesicular Body-Plasma Membrane Fusion and Exosome Release in Cultured Primary Neurons.

Exosomes represent a class of extracellular vesicles (EVs) derived from the endocytic pathway that is important for cell-cell communication and implicated in the spread of pathogenic protein aggregates associated with neurological diseases. Exosomes are released extracellularly when multivesicular bodies (also known as late endosomes) fuse with the plasma membrane (PM). An important breakthrough in exosome research is the ability to capture MVB-PM fusion and exosome release simultaneously in individual cells using live-imaging microscopy techniques. Specifically, researchers have created a construct fusing CD63, a tetraspanin enriched in exosomes, with the pH-sensitive reporter pHluorin whereby CD63-pHluorin fluorescence is quenched in the acidic MVB lumen and only fluoresces when released into the less acidic extracellular environment. Here, we describe a method using this CD63-pHluorin construct to visualize MVB-PM fusion/exosome secretion in primary neurons using total internal reflection fluorescence (TIRF) microscopy.

Culture of Primary Neurons from Dissociated and Cryopreserved Mouse Trigeminal Ganglion.

Corneal nerves originate from the ophthalmic branch of the trigeminal nerve, which enters the cornea at the limbus radially from all directions toward the central cornea. The cell bodies of the sensory neurons of trigeminal nerve are located in the trigeminal ganglion (TG), while the axons are extended into the three divisions, including ophthalmic branch that supplies corneal nerves. Study of primary neuronal cultures established from the TG fibers can therefore provide a knowledge basis for corneal nerve biology and potentially be developed as an in vitro platform for drug testing. However, setting up primary neuron cultures from animal TG has been dubious with inconsistency among laboratories due to a lack of efficient isolation protocol, resulting in low yield and heterogenous cultures. In this study, we used a combined enzymatic digestion with collagenase and TrypLE to dissociate mouse TG while preserving nerve cell viability. A subsequent discontinuous Percoll density gradient followed by mitotic inhibitor treatment effectively diminished the contamination of non-neuronal cells. Using this method, we reproducibly generated high yield and homogenous primary TG neuron cultures. Similar efficiency of nerve cell isolation and culture was further obtained for TG tissue cryopreserved for short (1 week) and long duration (3 months), compared to freshly isolated tissues. In conclusion, this optimized protocol shows a promising potential to standardize TG nerve culture and generate a high-quality corneal nerve model for drug testing and neurotoxicity studies.

Instant formation of horizontally ordered nanofibrous hydrogel films and direct investigation of peculiar neuronal cell behaviors atop.

BACKGROUND: Hydrogels have been widely used in many research fields owing to optical transparency, good biocompatibility, tunable mechanical properties, etc. Unlike typical hydrogels in the form of an unstructured bulk material, we developed aqueous dispersions of fiber-shaped hydrogel structures with high stability under ambient conditions and their application to various types of transparent soft cell culture interfaces with anisotropic nanoscale topography. METHOD: Nanofibers based on the polyvinyl alcohol and polyacrylic acid mixture were prepared by electrospinning and hydrogelified to nano-fibrous hydrogels (nFHs) after thermal crosslinking and sulfuric acid treatment. By modifying various material surfaces with positively-charged polymers, negatively-charged superabsorbent nFHs could be selectively patterned by employing micro-contact printing or horizontally aligned by applying shear force with a wired bar coater. RESULTS: The angular distribution of bar-coated nFHs was dramatically reduced to ± 20° along the applied shear direction unlike the drop-coated nFHs which exhibit random orientations. Next, various types of cells were cultured on top of transparent soft nFHs which showed good viability and attachment while their behaviors could be easily monitored by both upright and inverted optical microscopy. Particularly, neuronal lineage cells such as PC 12 cells and embryonic hippocampal neurons showed highly stretched morphology along the overall fiber orientation with aspect ratios ranging from 1 to 14. Furthermore, the resultant neurite outgrowth and migration behaviors could be effectively controlled by the horizontal orientation and the three-dimensional arrangement of underlying nFHs, respectively. CONCLUSION: We expect that surface modifications with transparent soft nFHs will be beneficial for various biological/biomedical studies such as fundamental cellular studies, neuronal/stem cell and/or organoid cultures, implantable probe/device coatings, etc.

Methods for shipping live primary cortical and hippocampal neuron cultures from postnatal mice.

Primary neuronal cultures have proven to be a powerful tool for studying mechanisms in neuroscience. It is technically challenging and expensive to reproduce high quality viable neuronal cultures. Laboratories that are not experienced or equipped to prepare primary neuron cultures may have difficulty producing consistent cultures for experiments. It has previously been shown that live rat embryonic hippocampal cultures can be shipped from laboratories that produce them. Here, we show that variations to this procedure allow for shipping postnatal mouse cultures of hippocampal and cortical primary neurons using standard commercial couriers. We also show that after shipping, primary neurons are viable, express synaptic markers, and demonstrate physiological activity, making them relevant models over immortalized cell lines. Among the many applications of this technique would be the preparation of cultured neurons from transgenic mouse lines in one laboratory and sharing them with distant collaborators, reducing variability.

Embelin prevents amyloid-beta accumulation via modulation of SOD1 in a Streptozotocin-induced AD-like condition: An evidence from in vitro investigation.

Embelin is a neuroprotective compound with therapeutic benefit against experimental Alzheimer's disease (AD)-like condition. In the quest of untangling the underlying mechanism behind the neuroprotective effect of Embelin in AD, an in-vitro study of Embelin against neuronal damage induced by Streptozotocin (STZ) in rat hippocampal neuronal culture was performed. Current findings demonstrated that Embelin (2.5-10 µM) has efficiently protected hippocampal neurons against STZ (8 mM)-induced neurotoxicity. An increase in amyloid precursor protein (APP), microtubule-associated protein tau (MAPT), glycogen synthase kinase 3 alpha (GSK-3α) and glycogen synthase kinase 3 beta (GSK-3ß) expression levels was observed when STZ (8 mM) stimulation was done for 24 h in the hippocampal neurons. A significant downregulation in the mRNA expression levels of APP, MAPT, GSK-3α, and GSK-3ß upon pre-treatment with different doses of Embelin (2.5 µM, 5 µM and 10 µM) reflects that Embelin attenuated STZ-induced dysfunction of insulin signaling (IR). Embelin significantly modulated the mRNA expression of scavenger enzyme Superoxide dismutase (SOD1). Furthermore, STZ had significantly upregulates an expression of Aß. On the contrary, pre-treatment with three doses of Embelin reversed an Aß-induced neuronal death. Our findings suggest that, Embelin prevents Aß accumulation via SOD1 pathway to protect against AD-like condition.

Spontaneous Cell Cluster Formation in Human iPSC-Derived Neuronal Spheroid Networks Influences Network Activity.

Three-dimensional neuronal culture systems such as spheroids, organoids, and assembloids constitute a branch of neuronal tissue engineering that has improved our ability to model the human brain in the laboratory. However, the more elaborate the brain model, the more difficult it becomes to study functional properties such as electrical activity at the neuronal level, similar to the challenges of studying neurophysiology in vivo We describe a simple approach to generate self-assembled three-dimensional neuronal spheroid networks with defined human cell composition on microelectrode arrays. Such spheroid networks develop a highly three-dimensional morphology with cell clusters up to 60 µm in thickness and are interconnected by pronounced bundles of neuronal fibers and glial processes. We could reliably record from up to hundreds of neurons simultaneously per culture for ≤90 d. By quantifying the formation of these three-dimensional structures over time, while regularly monitoring electrical activity, we were able to establish a strong link between spheroid morphology and network activity. In particular, the formation of cell clusters accelerates formation and maturation of correlated network activity. Astrocytes both influence electrophysiological network activity as well as accelerate the transition from single cell layers to cluster formation. Higher concentrations of astrocytes also have a strong effect of modulating synchronized network activity. This approach thus represents a practical alternative to often complex and heterogeneous organoids, providing easy access to activity within a brain-like 3D environment.Significance StatementNeuronal "organoid" cultures with multiple cell types grown on elaborate three-dimensional scaffolds have become popular tools to generate brain-like properties in vitro but bring with them similar problems concerning access to physiological function as real brain tissue. Here, we developed a new approach to form simple brain-like spheroid networks from human neurons, but using the normal supporting cells of the brain, astrocytes, as the scaffold. By growing these cultures on conventional microelectrode arrays, we were able to observe development of complex patterns of electrical activity for months. Our results highlight how formation of three-dimensional structures accelerated the formation of synchronized neuronal network activity and provide a promising new simple model system for studying interactions between known human cell types in vitro.

[Effective Viral Delivery of Genetic Constructs to Neuronal Culture for Modeling and Gene Therapy of GNAO1 Encephalopathy].

GNAO1 encephalopathy is an orphan genetic disease associated with early infantile epilepsy, impaired motor control, and severe developmental delay. The disorder is caused by mutations in the GNAO1 gene, leading to dysfunction of the encoded protein Gao1. There is no cure for this disease, and symptomatic therapy is ineffective. Phenotypic heterogeneity highlights the need for a personalized approach for treating patients with a specific clinical variant of GNAO1 and requires the study of the disease mechanism in animal and cell models. Towards this aim, we developed an approach for modeling GNAO1 encephalopathy and testing gene therapy drugs in primary neurons derived from healthy mice. We optimized the delivery of transgenes to Gαo1-expressing neurons using recombinant adeno-associated viruses (rAAV). We assessed the tropism of five neurotropic AAV serotypes (1, 2, 6, 9, DJ) for Gαo1-positive neurons from the whole mouse brain. The DJ serotype showed the highest potential as a reporter delivery vehicle, infecting up to 66% of Gαo1-expressing cells without overt cytotoxicity. We demonstrated that AAV-DJ also provides efficient delivery and expression of genetic constructs encoding normal and mutant Gαo1, as well as short hairpin RNA (shRNA) to suppress endogenous Gnao1 in murine neurons. Our results will further simplify the study of the pathological mechanism for clinical variants of GNAO1, as well as optimize the testing of gene therapy approaches for GNAO1 encephalopathy in cell models.

Protective role of VEGF/VEGFR2 signaling against high fatality associated with hepatic encephalopathy via sustaining mitochondrial bioenergetics functions.

BACKGROUND: The lack of better understanding of the pathophysiology and cellular mechanisms associated with high mortality seen in hepatic encephalopathy (HE), a neurological complication arising from acute hepatic failure, remains a challenging medical issue. Clinical reports showed that the degree of baroreflex dysregulation is related to the severity of HE. Furthermore, mitochondrial dysfunction in the rostral ventrolateral medulla (RVLM), a key component of the baroreflex loop that maintains blood pressure and sympathetic vasomotor tone, is known to underpin impairment of baroreflex. Realizing that in addition to angiogenic and vasculogenic effects, by acting on its key receptor (VEGFR2), vascular endothelial growth factor (VEGF) elicits neuroprotection via maintenance of mitochondrial function, the guiding hypothesis of the present study is that the VEGF/VEGFR2 signaling plays a protective role against mitochondrial dysfunction in the RVLM to ameliorate baroreflex dysregulation that underpins the high fatality associated with HE. METHODS: Physiological, pharmacological and biochemical investigations were carried out in proof-of-concept experiments using an in vitro model of HE that involved incubation of cultured mouse hippocampal neurons with ammonium chloride. This was followed by corroboratory experiments employing a mouse model of HE, in which adult male C57BL/6 mice and VEGFR2 wild-type and heterozygous mice received an intraperitoneal injection of azoxymethane, a toxin used to induce acute hepatic failure. RESULTS: We demonstrated that VEGFR2 is present in cultured neurons, and observed that whereas recombinant VEGF protein maintained cell viability, gene-knockdown of vegfr2 enhanced the reduction of cell viability in our in vitro model of HE. In our in vivo model of HE, we found that VEGFR2 heterozygous mice exhibited shorter survival rate and time when compared to wild-type mice. In C57BL/6 mice, there was a progressive reduction in VEGFR2 mRNA and protein expression, mitochondrial membrane potential and ATP levels, alongside augmentation of apoptotic cell death in the RVLM, accompanied by a decrease in baroreflex-mediated sympathetic vasomotor tone and hypotension. Immunoneutralization of VEGF exacerbated all those biochemical and physiological events. CONCLUSIONS: Our results suggest that, acting via VEGFR2, the endogenous VEGF plays a protective role against high fatality associated with HE by amelioration of the dysregulated baroreflex-mediated sympathetic vasomotor tone through sustaining mitochondrial bioenergetics functions and eliciting antiapoptotic action in the RVLM.

Optogenetic Control of Human Stem Cell-Derived Neurons.

Spontaneous and optogenetically evoked activities of human induced pluripotent stem cell (hiPSC)-derived neurons can be assessed by patch clamp and multi-electrode array (MEA) electrophysiology. Optogenetic activation of these human neurons facilitates the characterization of their functional properties at the single neuron and circuit level. Here we showcase the preparation of hiPSC-derived neurons expressing optogenetic actuators, in vitro optogenetic stimulation and simultaneous functional recordings using patch clamp and MEA electrophysiology.

Multiplexing Seahorse XFe24 and ImageXpress® Nano Platforms for Comprehensive Evaluation of Mitochondrial Bioenergetic Profile and Neuronal Morphology.

The measurement of mitochondrial function has become imperative to understand and characterize diseases characterized by bioenergetic alterations. The advancement of automation and application of high-throughput technologies has propelled our understanding of biological complexity and facilitated drug discovery. Seahorse extracellular flux (XFe) technology measures changes in dissolved oxygen and proton concentration in cell culture media, providing kinetic measurements of oxidative phosphorylation and glycolytic metabolism. ImageXpress® Nano is an automated fluorescent microscope with the ability to perform high-content, fast, and robust imaging in multi-well formats. In this chapter, we present a comprehensive protocol to multiplex the Seahorse XFe24 analyzer with the ImageXpress® Nano high content imaging microscope to provide a comprehensive yet rigorous profile of bioenergetics and its correlation to neuronal function and morphology.

1, 25-(OH)2D3 protects against ER stress and miRNA dysregulation in Mus musculus neurons.

BACKGROUND: The pathophysiology of neurodegenerative diseases (NDDs) is closely associated with cellular oxidative stress which can result in the accumulation of toxic proteins in the endoplasmic reticulum (ER) leading to ER stress and subsequent unfolded protein response (UPR) signaling, a mechanism that aggravate these disorders. Vitamin D has been suggested to have important neuroprotective role and its administration has been shown to reduce neuronal injury, neurotoxicity and oxidative stress in various animal systems. OBJECTIVE: The current study was undertaken to examine the effect of vitamin D3 on UPR in ER stress induced Mus musculus neuronal cells. METHODS: Mus musculus cortical and hippocampal primary neuronal cultures were pretreated with 1,25-dihydroxyvitamin D3 (1, 25-(OH)2D3), the active form of vitamin D, followed by ER stress induction with a chemical ER stress inducer thapsigargin and with an advanced glycated protein, AGE-BSA. The UPR genes and related microRNAs (miRNA) expressions were analyzed mainly using real-time PCR. RESULTS: The experiment resulted in the suppression of ER stress marker BiP and UPR pathway genes such as Perk, Ire1α, Chop and Puma which mediate cellular apoptosis indicating the protective effect of 1, 25-(OH)2D3 against neuronal ER stress. Further studies into the molecular aspects showed that ER stress mediated down-regulated expression of microRNAs (miRNAs) such as mmu-miR-24, 27b, 124, 224, 290, 351 and 488 which are known to regulate the UPR pathway genes were also reduced with vitamin pretreatment, of which the miRNAs miR-24 and 27b which shares the same cluster are potentially involved in various human diseases. CONCLUSION: This study emphasizes the therapeutic role of vitamin D in reducing neuronal ER stress and the need for maintaining sufficient amount of this vitamin in our diet.