Disease-associated polyalanine expansion mutations impair UBA6-dependent ubiquitination.

Expansion mutations in polyalanine stretches are associated with a growing number of diseases sharing a high degree of genotypic and phenotypic commonality. These similarities prompted us to query the normal function of physiological polyalanine stretches and to investigate whether a common molecular mechanism is involved in these diseases. Here, we show that UBA6, an E1 ubiquitin-activating enzyme, recognizes a polyalanine stretch within its cognate E2 ubiquitin-conjugating enzyme USE1. Aberrations in this polyalanine stretch reduce ubiquitin transfer to USE1 and, subsequently, polyubiquitination and degradation of its target, the ubiquitin ligase E6AP. Furthermore, we identify competition for the UBA6-USE1 interaction by various proteins with polyalanine expansion mutations in the disease state. The deleterious interactions of expanded polyalanine tract proteins with UBA6 in mouse primary neurons alter the levels and ubiquitination-dependent degradation of E6AP, which in turn affects the levels of the synaptic protein Arc. These effects are also observed in induced pluripotent stem cell-derived autonomic neurons from patients with polyalanine expansion mutations, where UBA6 overexpression increases neuronal resilience to cell death. Our results suggest a shared mechanism for such mutations that may contribute to the congenital malformations seen in polyalanine tract diseases.

Acoustofluidic Engineering of Functional Vessel-on-a-Chip.

Construction of in vitro vascular models is of great significance to various biomedical research, such as pharmacokinetics and hemodynamics, and thus is an important direction in the tissue engineering field. In this work, a standing surface acoustic wave field was constructed to spatially arrange suspended endothelial cells into a designated acoustofluidic pattern. The cell patterning was maintained after the acoustic field was withdrawn within the solidified hydrogel. Then, interstitial flow was provided to activate vessel tube formation. In this way, a functional vessel network with specific vessel geometry was engineered on-chip. Vascular function, including perfusability and vascular barrier function, was characterized by microbead loading and dextran diffusion, respectively. A computational atomistic simulation model was proposed to illustrate how solutes cross the vascular membrane lipid bilayer. The reported acoustofluidic methodology is capable of facile and reproducible fabrication of the functional vessel network with specific geometry and high resolution. It is promising to facilitate the development of both fundamental research and regenerative therapy.

Brain-Targeted Liposomes Loaded with Monoclonal Antibodies Reduce Alpha-Synuclein Aggregation and Improve Behavioral Symptoms in Parkinson's Disease.

Monoclonal antibodies (mAbs) hold promise in treating Parkinson's disease (PD), although poor delivery to the brain hinders their therapeutic application. In the current study, it is demonstrated that brain-targeted liposomes (BTL) enhance the delivery of mAbs across the blood-brain-barrier (BBB) and into neurons, thereby allowing the intracellular and extracellular treatment of the PD brain. BTL are decorated with transferrin to improve brain targeting through overexpressed transferrin-receptors on the BBB during PD. BTL are loaded with SynO4, a mAb that inhibits alpha-synuclein (AS) aggregation, a pathological hallmark of PD. It is shown that 100-nm BTL cross human BBB models intact and are taken up by primary neurons. Within neurons, SynO4 is released from the nanoparticles and bound to its target, thereby reducing AS aggregation, and enhancing neuronal viability. In vivo, intravenous BTL administration results in a sevenfold increase in mAbs in brain cells, decreasing AS aggregation and neuroinflammation. Treatment with BTL also improve behavioral motor function and learning ability in mice, with a favorable safety profile. Accordingly, targeted nanotechnologies offer a valuable platform for drug delivery to treat brain neurodegeneration.

Acoustofluidic Engineering Functional Vessel-on-a-Chip.

Construction of in vitro vascular models is of great significance to various biomedical research, such as pharmacokinetics and hemodynamics, thus is an important direction in tissue engineering. In this work, a standing surface acoustic wave field was constructed to spatially arrange suspended endothelial cells into a designated patterning. The cell patterning was maintained after the acoustic field was withdrawn by the solidified hydrogel. Then, interstitial flow was provided to activate vessel tube formation. Thus, a functional vessel-on-a-chip was engineered with specific vessel geometry. Vascular function, including perfusability and vascular barrier function, was characterized by beads loading and dextran diffusion, respectively. A computational atomistic simulation model was proposed to illustrate how solutes cross vascular lipid bilayer. The reported acoustofluidic methodology is capable of facile and reproducible fabrication of functional vessel network with specific geometry. It is promising to facilitate the development of both fundamental research and regenerative therapy.

Oculopharyngeal muscular dystrophy mutations link the RNA-binding protein HNRNPQ to autophagosome biogenesis.

Autophagy is an intracellular degradative process with an important role in cellular homeostasis. Here, we show that the RNA binding protein (RBP), heterogeneous nuclear ribonucleoprotein Q (HNRNPQ)/SYNCRIP is required to stimulate early events in autophagosome biogenesis, in particular the induction of VPS34 kinase by ULK1-mediated beclin 1 phosphorylation. The RBPs HNRNPQ and poly(A) binding protein nuclear 1 (PABPN1) form a regulatory network that controls the turnover of distinct autophagy-related (ATG) proteins. We also show that oculopharyngeal muscular dystrophy (OPMD) mutations engender a switch from autophagosome stimulation to autophagosome inhibition by impairing PABPN1 and HNRNPQ control of the level of ULK1. The overexpression of HNRNPQ in OPMD patient-derived cells rescues the defective autophagy in these cells. Our data reveal a regulatory mechanism of autophagy induction that is compromised by PABPN1 disease mutations, and may thus further contribute to their deleterious effects.

3D models of glioblastoma interaction with cortical cells.

Introduction: Glioblastoma (GBM) invasiveness and ability to infiltrate deep into the brain tissue is a major reason for the poor patient prognosis for this type of brain cancer. Behavior of glioblastoma cells, including their motility, and expression of invasion-promoting genes such as matrix metalloprotease-2 (MMP2), are strongly influenced by normal cells found in the brain parenchyma. Cells such as neurons may also be influenced by the tumor, as many glioblastoma patients develop epilepsy. In vitro models of glioblastoma invasiveness are used to supplement animal models in a search for better treatments, and need to combine capability for high-throughput experiments with capturing bidirectional interactions between GBM and brain cells. Methods: In this work, two 3D in vitro models of GBM-cortical interactions were investigated. A matrix-free model was created by co-culturing GBM and cortical spheroids, and a matrix-based model was created by embedding cortical cells and a GBM spheroid in Matrigel. Results: Rapid GBM invasion occurred in the matrix-based model, and was enhanced by the presence of cortical cells. Little invasion occurred in the matrix-free model. In both types of models, presence of GBM cells resulted in a significant increase in paroxysmal neuronal activity. Discussion: Matrix-based model may be better suited for studying GBM invasion in an environment that includes cortical cells, while matrix-free model may be useful in investigation of tumor-associated epilepsy.

Efficient and Accurate Computational Model of Neuron with Spike Frequency Adaptation.

Simplified models of neurons are widely used in computational investigations of large networks. One of the most important performance metrics of simplified models is their accuracy in reproducing action potential (spike) timing. In this article, we developed a simple, computationally efficient neuron model by modifying the adaptive exponential integrate and fire (AdEx) model [1] with sigmoid afterhyperpolarization current (Sigmoid AHP). Our model can precisely match the spike times and spike frequency adaptation of cortical pyramidal neurons. The accuracy was similar to a more complex two compartment biophysically realistic model of the same neurons. This work provides a simplified neuronal model with improved spike timing accuracy for use in modeling of large neural networks.Clinical Relevance- Accurate and computationally efficient single neuron model will enable large network modeling of brain regions involved in neurological and psychiatric disorders and may lead to a better understanding of the disorder mechanisms.

Microdevice for directional axodendritic connectivity between micro 3D neuronal cultures.

Neuronal cultures are widely used in neuroscience research. However, the randomness of circuits in conventional cultures prevents accurate in vitro modeling of cortical development and of the pathogenesis of neurological and psychiatric disorders. A basic feature of cortical circuits that is not captured in standard cultures of dissociated cortical cells is directional connectivity. In this work, a polydimethylsiloxane (PDMS)-based device that achieves directional connectivity between micro 3D cultures is demonstrated. The device consists of through-holes for micro three-dimensional (µ3D) clusters of cortical cells connected by microtrenches for axon and dendrite guidance. The design of the trenches relies in part on the concept of axonal edge guidance, as well as on the novel concept of specific dendrite targeting. This replicates dominant excitatory connectivity in the cortex, enables the guidance of the axon after it forms a synapse in passing (an "en passant" synapse), and ensures that directional selectivity is preserved over the lifetime of the culture. The directionality of connections was verified morphologically and functionally. Connections were dependent on glutamatergic synapses. The design of this device has the potential to serve as a building block for the reconstruction of more complex cortical circuits in vitro.

Understanding the Impact of Neural Variations and Random Connections on Inference.

Recent research suggests that in vitro neural networks created from dissociated neurons may be used for computing and performing machine learning tasks. To develop a better artificial intelligent system, a hybrid bio-silicon computer is worth exploring, but its performance is still inferior to that of a silicon-based computer. One reason may be that a living neural network has many intrinsic properties, such as random network connectivity, high network sparsity, and large neural and synaptic variability. These properties may lead to new design considerations, and existing algorithms need to be adjusted for living neural network implementation. This work investigates the impact of neural variations and random connections on inference with learning algorithms. A two-layer hybrid bio-silicon platform is constructed and a five-step design method is proposed for the fast development of living neural network algorithms. Neural variations and dynamics are verified by fitting model parameters with biological experimental results. Random connections are generated under different connection probabilities to vary network sparsity. A multi-layer perceptron algorithm is tested with biological constraints on the MNIST dataset. The results show that a reasonable inference accuracy can be achieved despite the presence of neural variations and random network connections. A new adaptive pre-processing technique is proposed to ensure good learning accuracy with different living neural network sparsity.

Neuron and astrocyte aggregation and sorting in three-dimensional neuronal constructs.

Aggregation and self-sorting of cells in three dimensional cultures have been described for non-neuronal cells. Despite increased interest in engineered neural tissues for treating brain injury or for modeling neurological disorders in vitro, little data is available on collective cell movements in neuronal aggregates. Migration and sorting of cells may alter these constructs' morphology and, therefore, the function of their neural circuitry. In this work, linear, adhered rat and human 3D neuronal-astrocyte cultures were developed to enable the study of aggregation and sorting of these cells. An in silico model of the contraction, clustering, and cell sorting in the 3D cultures was also developed. Experiments and computational modeling showed that aggregation was mainly a neuron mediated process, and formation of astrocyte-rich sheaths in 3D cultures depended on differential attraction between neurons and astrocytes. In silico model predicted formation of self-assembled neuronal layers in disk-shaped 3D cultures. Neuronal activity patterns were found to correlate with local morphological differences. This model of neuronal and astrocyte aggregation and sorting may benefit future design of neuronal constructs.

The molecular basis of Rac-GTP action-promoting binding of p67phox to Nox2 by disengaging the ß hairpin from downstream residues.

p67phox fulfils a key role in the assembly/activation of the NADPH oxidase by direct interaction with Nox2. We proposed that Rac-GTP serves both as a carrier of p67phox to the membrane and an inducer of a conformational change enhancing its affinity for Nox2. This study provides evidence for the latter function: (i) oxidase activation was inhibited by p67phox peptides (106-120) and (181-195), corresponding to the ß hairpin and to a downstream region engaged in intramolecular bonds with the ß hairpin, respectively; (ii) deletion of residues 181-193 and point mutations Q115R or K181E resulted in selective binding of p67phox to Nox2 peptide (369-383); (iii) both deletion and point mutations led to a change in p67phox , expressed in increased apparent molecular weights; (iv) p67phox was bound to p67phox peptide (181-195) and to a cluster of peptides (residues 97-117), supporting the participation of selected residues within these sequences in intramolecular bonds; (v) p67phox failed to bind to Nox2 peptide (369-383), following interaction with Rac1-GTP, but a (p67phox -Rac1-GTP) chimera exhibited marked binding to the peptide, similar to that of p67phox deletion and point mutants; and (vi) size exclusion chromatography of the chimera revealed its partition in monomeric and polymeric forms, with binding to Nox2 peptide (369-383) restricted to polymers. The molecular basis of Rac-GTP action entails unmasking of a previously hidden Nox2-binding site in p67phox , following disengagement of the ß hairpin from more C-terminal residues. The domain in Nox2 binding the "modified" p67phox comprises residues within the 369-383 sequence in the cytosolic dehydrogenase region.

Deubiquitylating enzymes in neuronal health and disease.

Ubiquitylation and deubiquitylation play a pivotal role in protein homeostasis (proteostasis). Proteostasis shapes the proteome landscape in the human brain and its impairment is linked to neurodevelopmental and neurodegenerative disorders. Here we discuss the emerging roles of deubiquitylating enzymes in neuronal function and survival. We provide an updated perspective on the genetics, physiology, structure, and function of deubiquitylases in neuronal health and disease.

p67phox -derived self-assembled peptides prevent Nox2 NADPH oxidase activation by an auto-inhibitory mechanism.

Activation of the Nox2-dependent NADPH oxidase is the result of a conformational change in Nox2 induced by interaction with the cytosolic component p67phox . In preliminary work we identified a cluster of overlapping 15-mer synthetic peptides, corresponding to p67phox residues 259-279, which inhibited oxidase activity in an in vitro, cell-free assay, but the results did not point to a competitive mechanism. We recently identified an auto-inhibitory intramolecular bond in p67phox , one extremity of which was located within the 259-279 sequence, and we hypothesized that inhibition by exogenous peptides might mimic intrinsic auto-inhibition. In this study, we found that: (i) progressive N- and C-terminal truncation of inhibitory p67phox peptides, corresponding to residues 259-273 and 265-279, revealed that inhibitory ability correlated with the presence of residues 265 NIVFVL270 , exposed at either the N- or C-termini of the peptides; (ii) inhibition of oxidase activity was associated exclusively with self-assembled peptides, which pelleted upon centrifugation at 12,000 ×g; (iii) self-assembled p67phox peptides inhibited oxidase activity by specific binding of p67phox and the ensuing depletion of this component, essential for interaction with Nox2; and (iv) peptides subjected to scrambling or reversing the order of residues in NIVFVL retained the propensity for self-assembly, oxidase inhibitory ability, and specific binding of p67phox , indicating that the dominant parameter was the hydrophobic character of five of the six residues. This appears to be the first description of inhibition of oxidase activity by self-assembled peptides derived from an oxidase component, acting by an auto-inhibitory mechanism.

Variability of seizure-like activity in an in vitro model of epilepsy depends on the electrical recording method.

BACKGROUND: Hippocampal and cortical slice-based models are widely used to study seizures and epilepsy. Seizure detection and quantification are essential components for studying mechanisms of epilepsy and assessing therapeutic interventions. To obtain meaningful signals and maximize experimental throughput, variability should be minimized. Some electrical recording methods require insertion of an electrode into neuronal tissue, change in slice chemical microenvironment, and transients in temperature and pH. These perturbations can cause acute and long-term alterations of the neuronal network which may be reflected in the variability of the recorded signal. NEW METHOD: In this study we investigated the effect of experimental perturbations in three local field potential (LFP) recording methods including substrate micro-wires (s-MWs), multiple electrode arrays (MEAs), and inserted micro wire electrodes (i-MW). These methods enabled us to isolate effects of different perturbations. We used organotypic hippocampal slices (OHCs) as an in-vitro model of posttraumatic epilepsy. To investigate the effect of the disturbances caused by the recording method on the paroxysmal events, we introduced jitter analysis, which is sensitive to small differences in the seizure spike timing. RESULTS: Medium replacement can introduce long-lasting perturbations. Electrode insertion increased variability on a shorter time scale. OHCs also underwent spontaneous state transitions characterized by transient increases in variability. COMPARISON WITH EXISTING METHODS: This new method of seizure waveform analysis allows for more sensitive assessment of variability of ictal events than simply measuring seizure frequency and duration. CONCLUSION: We demonstrated that some of the variability in OHC recordings are due to experimental perturbations while some are spontaneous and independent of recording method.

Micro Three-Dimensional Neuronal Cultures Generate Developing Cortex-Like Activity Patterns.

Studies aimed at neurological drug discovery have been carried out both in vitro and in vivo. In vitro cell culture models have showed potential as drug testing platforms characterized by high throughput, low cost, good reproducibility and ease of handling and observation. However, in vitro neuronal culture models are facing challenges in replicating in vivo-like activity patterns. This work reports an in vitro culture technique that is capable of producing micro three-dimensional (µ3D) cultures of only a few tens of neurons. The µ3D cultures generated by this method were uniform in size and density of neurons. These µ3D cultures had complex spontaneous synchronized neuronal activity patterns which were similar to those observed in the developing cortex and in much larger 3D cultures, but not in 2D cultures. Bursts could be reliably evoked by stimulation of single neurons. Synchronized bursts in µ3D cultures were abolished by inhibitors of glutamate receptors, while inhibitors of GABAA receptors had a more complex effect. This pharmacological profile is similar to bursts in neonatal cortex. Since large numbers of reproducible µ3D cultures can be created and observed in parallel, this model of the developing cortex may find applications in high-throughput drug discovery experiments.

p67phox binds to a newly identified site in Nox2 following the disengagement of an intramolecular bond-Canaan sighted?

Activation of the phagocyte NADPH oxidase involves a conformational change in Nox2. The effector in this process is p67phox and there is evidence for a change in the configuration of p67phox being required for binding to Nox2. To study this, we measured binding of p67phox to a library of Nox2 peptides and binding of NusA-Nox2 fusion proteins to p67phox . We found, serendipitously, that deletion of residues 259-279 in p67phox (p67phox Δ(259-279)), endowed it with the ability to bind selectively to Nox2 peptide 369-383 (peptide 28). There was no binding to scrambled Nox2 peptide 28 and to Nox4 peptide 28. Binding was cysteine independent and resistant to reducing and alkylating agents. Truncations of peptide 28 revealed that the actual binding site consisted of residues 375-383. Binding of p67phox Δ(259-279) to peptide 28 was mimicked by that of a (p67phox -RacGTP) chimera. Both p67phox Δ(259-279) and the (p67pho -RacGTP) chimera bound a NusA-Nox2 fusion protein, comprising residues 375-383. Specific single residue deletion mutants, within the p67phox sequence 259-279, were also bound to Nox2 peptide 28. Peptides synthesized to correspond to the 259-279 sequence in p67phox , were found to autobind p67phox , suggesting that an intramolecular bond exists in p67phox , one pole of which was located within residues 259-279. We conclude that "resting" p67phox exists in a "closed" conformation, generated by an intramolecular bond. Deletion of specific residues within the 259-279 sequence, in vitro, or interaction with RacGTP, in vivo, causes "opening" of the bond and results in binding of p67phox to a specific, previously unknown, site in Nox2.

Neural layer self-assembly in geometrically confined rat and human 3D cultures.

Neurological disorders affect millions of Americans and this number is expected to rise with the aging population. Development of drugs to treat these disorders may be facilitated by improved in vitro models that faithfully reproduce salient features of the relevant brain regions. Current 3D culture methods face challenges with reliably reproducing microarchitectural features of brain morphology such as cortical or hippocampal layers. In this work, polydimethylsiloxane (PDMS) mini-wells were used to create low aspect ratio, adherent 3D constructs where neurons self-assemble into layers. Layer self-assembly was determined to depend on the size of the PDMS mini-well. Layer formation occurred in cultures composed of primary rat cortical neurons or human induced pluripotent stem cell-derived neurons and astrocytes and was robust and reproducible. Layered 3D constructs were found to have spontaneous neural activity characterized by long bursts similar to activity in the developing cortex. The responses of layered 3D cultures to drugs were more similar to in vivo data than those of 2D cultures. 3D constructs created with this method may be thus suitable as in vitro models for drug discovery for neurological disorders.

Kinase Inhibitors with Antiepileptic Properties Identified with a Novel in Vitro Screening Platform.

Kinase signaling plays an important role in acquired epilepsy, but only a small percentage of the total kinome has been investigated in this context. A major roadblock that prevents the systematic investigation of the contributions of kinase signaling networks is the slow speed of experiments designed to test the chronic effects of target inhibition in epilepsy models. We developed a novel in vitro screening platform based on microwire recordings from an organotypic hippocampal culture model of acquired epilepsy. This platform enables the direct, parallel determination of the effects of compounds on spontaneous epileptiform activity. The platform also enables repeated recordings from the same culture over two-week long experiments. We screened 45 kinase inhibitors and quantified their effects on seizure duration, the frequency of paroxysmal activity, and electrographic load. We identified several inhibitors with previously unknown antiepileptic properties. We also used kinase inhibition profile cross-referencing to identify kinases that are inhibited by seizure-suppressing compounds, but not by compounds that had no effect on seizures.

Epilepsy-on-a-Chip System for Antiepileptic Drug Discovery.

OBJECTIVE: Hippocampal slice cultures spontaneously develop chronic epilepsy several days after slicing and are used as an in vitro model of post-traumatic epilepsy. Here, we describe a hybrid microfluidic-microelectrode array (µflow-MEA) technology that incorporates a microfluidic perfusion network and electrodes into a miniaturized device for hippocampal slice culture based antiepileptic drug discovery. METHODS: Field potential simulation was conducted to help optimize the electrode design to detect a seizure-like population activity. Epilepsy-on-a-chip model was validated by chronic electrical recording, neuronal survival quantification, and anticonvulsant test. To demonstrate the application of µflow-MEA in drug discovery, we utilized a two-stage screening platform to identify potential targets for antiepileptic drugs. In Stage I, lactate and lactate dehydrogenase biomarker assays were performed to identify potential drug candidates. In Stage II, candidate compounds were retested with µflow-MEA-based chronic electrical assay to provide electrophysiological confirmation of biomarker results. RESULTS AND CONCLUSION: We screened 12 receptor tyrosine kinases inhibitors, and EGFR/ErbB-2 and cFMS inhibitors were identified as novel antiepileptic compounds. SIGNIFICANCE: This epilepsy-on-a-chip system provides the means for rapid dissection of complex signaling pathways in epileptogenesis, paving the way for high-throughput antiepileptic drug discovery.