Increasing glutathione levels by a novel posttranslational mechanism inhibits neuronal hyperexcitability.

Glutathione (GSH) depletion, and impaired redox homeostasis have been observed in experimental animal models and patients with epilepsy. Pleiotropic strategies that elevate GSH levels via transcriptional regulation have been shown to significantly decrease oxidative stress and seizure frequency, increase seizure threshold, and rescue certain cognitive deficits. Whether elevation of GSH per se alters neuronal hyperexcitability remains unanswered. We previously showed that thiols such as dimercaprol (DMP) elevate GSH via post-translational activation of glutamate cysteine ligase (GCL), the rate limiting GSH biosynthetic enzyme. Here, we asked if elevation of cellular GSH by DMP altered neuronal hyperexcitability in-vitro and in-vivo. Treatment of primary neuronal-glial cerebrocortical cultures with DMP elevated GSH and inhibited a voltage-gated potassium channel blocker (4-aminopyridine, 4AP) induced neuronal hyperexcitability. DMP increased GSH in wildtype (WT) zebrafish larvae and significantly attenuated convulsant pentylenetetrazol (PTZ)-induced acute 'seizure-like' swim behavior. DMP treatment increased GSH and inhibited convulsive, spontaneous 'seizure-like' swim behavior in the Dravet Syndrome (DS) zebrafish larvae (scn1Lab). Furthermore, DMP treatment significantly decreased spontaneous electrographic seizures and associated seizure parameters in scn1Lab zebrafish larvae. We investigated the role of the redox-sensitive mammalian target of rapamycin (mTOR) pathway due to the presence of several cysteine-rich proteins and their involvement in regulating neuronal excitability. Treatment of primary neuronal-glial cerebrocortical cultures with 4AP or l-buthionine-(S,R)-sulfoximine (BSO), an irreversible inhibitor of GSH biosynthesis, significantly increased mTOR complex I (mTORC1) activity which was rescued by pre-treatment with DMP. Furthermore, BSO-mediated GSH depletion oxidatively modified the tuberous sclerosis protein complex (TSC) consisting of hamartin (TSC1), tuberin (TSC2), and TBC1 domain family member 7 (TBC1D7) which are critical negative regulators of mTORC1. In summary, our results suggest that DMP-mediated GSH elevation by a novel post-translational mechanism can inhibit neuronal hyperexcitability both in-vitro and in-vivo and a plausible link is the redox sensitive mTORC1 pathway.

Neuronal SIRT3 Deletion Predisposes to Female-Specific Alterations in Cellular Metabolism, Memory, and Network Excitability.

Mitochondrial dysfunction is an early event in the pathogenesis of neurologic disorders and aging. Sirtuin 3 (SIRT3) regulates mitochondrial function in response to the cellular environment through the reversible deacetylation of proteins involved in metabolism and reactive oxygen species detoxification. As the primary mitochondrial deacetylase, germline, or peripheral tissue-specific deletion of SIRT3 produces mitochondrial hyperacetylation and the accelerated development of age-related diseases. Given the unique metabolic demands of neurons, the role of SIRT3 in the brain is only beginning to emerge. Using mass spectrometry-based acetylomics, high-resolution respirometry, video-EEG, and cognition testing, we report targeted deletion of SIRT3 from select neurons in the cortex and hippocampus produces altered neuronal excitability and metabolic dysfunction in female mice. Targeted deletion of SIRT3 from neuronal helix-loop-helix 1 (NEX)-expressing neurons resulted in mitochondrial hyperacetylation, female-specific superoxide dismutase-2 (SOD2) modification, increased steady-state superoxide levels, metabolic reprogramming, altered neuronal excitability, and working spatial memory deficits. Inducible neuronal deletion of SIRT3 likewise produced female-specific deficits in spatial working memory. Together, the data demonstrate that deletion of SIRT3 from forebrain neurons selectively predisposes female mice to deficits in mitochondrial and cognitive function.SIGNIFICANCE STATEMENT Mitochondrial SIRT3 is an enzyme shown to regulate energy metabolism and antioxidant function, by direct deacetylation of proteins. In this study, we show that neuronal SIRT3 deficiency renders female mice selectively vulnerable to impairment in redox and metabolic function, spatial memory, and neuronal excitability. The observed sex-specific effects on cognition and neuronal excitability in female SIRT3-deficient mice suggest that mitochondrial dysfunction may be one factor underlying comorbid neuronal diseases, such as Alzheimer's disease and epilepsy. Furthermore, the data suggest that SIRT3 dysfunction may predispose females to age-related metabolic and cognitive impairment.

Nutrient-sensing mTORC1 and AMPK pathways in chronic kidney diseases.

Nutrients such as glucose, amino acids and lipids are fundamental sources for the maintenance of essential cellular processes and homeostasis in all organisms. The nutrient-sensing kinases mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are expressed in many cell types and have key roles in the control of cell growth, proliferation, differentiation, metabolism and survival, ultimately contributing to the physiological development and functions of various organs, including the kidney. Dysregulation of these kinases leads to many human health problems, including cancer, neurodegenerative diseases, metabolic disorders and kidney diseases. In the kidney, physiological levels of mTOR and AMPK activity are required to support kidney cell growth and differentiation and to maintain kidney cell integrity and normal nephron function, including transport of electrolytes, water and glucose. mTOR forms two functional multi-protein kinase complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Hyperactivation of mTORC1 leads to podocyte and tubular cell dysfunction and vulnerability to injury, thereby contributing to the development of chronic kidney diseases, including diabetic kidney disease, obesity-related kidney disease and polycystic kidney disease. Emerging evidence suggests that targeting mTOR and/or AMPK could be an effective therapeutic approach to controlling or preventing these diseases.

Neuron-specific mitochondrial oxidative stress results in epilepsy, glucose dysregulation and a striking astrocyte response.

Mitochondrial superoxide (O2-) production is implicated in aging, neurodegenerative disease, and most recently epilepsy. Yet the specific contribution of neuronal O2- to these phenomena is unclear. Here, we selectively deleted superoxide dismutase-2 (SOD2) in neuronal basic helix-loop-helix transcription factor (NEX)-expressing cells restricting deletion to a subset of excitatory principle neurons primarily in the forebrain (cortex and hippocampus). This resulted in nSOD2 KO mice that lived into adulthood (2-3 months) with epilepsy, selective loss of neurons, metabolic rewiring and a marked mitohormetic gene response. Surprisingly, expression of an astrocytic gene, glial fibrillary acidic protein (GFAP) was significantly increased relative to WT. Further studies in rat primary neuron-glial cultures showed that increased mitochondrial O2-, specifically in neurons, was sufficient to upregulate GFAP. These results suggest that neuron-specific mitochondrial O2- is sufficient to drive a complex and catastrophic epileptic phenotype and highlights the ability of SOD2 to act in a cell-nonautonomous manner to influence an astrocytic response.

Enhancing glucose metabolism via gluconeogenesis is therapeutic in a zebrafish model of Dravet syndrome.

Energy-producing pathways are novel therapeutic targets for the treatment of neurodevelopmental disorders. Here, we focussed on correcting metabolic defects in a catastrophic paediatric epilepsy, Dravet syndrome which is caused by mutations in sodium channel NaV1.1 gene, SCN1A. We utilized a translatable zebrafish model of Dravet syndrome (scn1lab) which exhibits key characteristics of patients with Dravet syndrome and shows metabolic deficits accompanied by down-regulation of gluconeogenesis genes, pck1 and pck2. Using a metabolism-based small library screen, we identified compounds that increased gluconeogenesis via up-regulation of pck1 gene expression in scn1lab larvae. Treatment with PK11195, a pck1 activator and a translocator protein ligand, normalized dys-regulated glucose levels, metabolic deficits, translocator protein expression and significantly decreased electrographic seizures in mutant larvae. Inhibition of pck1 in wild-type larvae mimicked metabolic and behaviour defects observed in scn1lab mutants. Together, this suggests that correcting dys-regulated metabolic pathways can be therapeutic in neurodevelopmental disorders such as Dravet syndrome arising from ion channel dysfunction.

Mitochondrial transfer from mesenchymal stem cells improves neuronal metabolism after oxidant injury in vitro: The role of Miro1.

Stroke-induced cerebral ischemia is a major cause of death and disability. The disruption of blood flow results in neuronal and glial cell death leading to brain injury. Reperfusion restores oxygen to the affected tissue, but can also cause damage through an enhanced oxidative stress and inflammatory response. This study examines mitochondrial transfer from MSC to neurons and the role it plays in neuronal preservation after oxidant injury. We observed the transfer of mitochondria from MSC to mouse neurons in vitro following hydrogen peroxide exposure. The observed transfer was dependent on cell-to-cell contact and led to increased neuronal survival and improved metabolism. A number of pro-inflammatory and mitochondrial motility genes were upregulated in neurons after hydrogen peroxide exposure. This included Miro1 and TNFAIP2, linking inflammation and mitochondrial transfer to oxidant injury. Increasing Miro1 expression in MSC improved the metabolic benefit of mitochondrial transfer after neuronal oxidant injury. Decreasing Miro1 expression had the opposite effect, decreasing the metabolic benefit of MSC co-culture. MSC transfer of mitochondria to oxidant-damaged neurons may help improve neuronal preservation and functional recovery after stroke.

Bicuspid Aortic Valve in Pregnancy Complicated by Aortic Valve Vegetation, Aortic Root Abscess, and Aortic Insufficiency.

A 39-year-old patient presented to our Labor and Delivery unit with fever and nausea in the context of recent bacteriuria. She was found to be in sepsis due to an incidental bicuspid aortic valve (BAV) complicated by aortic valvular vegetations, severe aortic insufficiency, and aortic root abscess, requiring an emergent cesarean section. Three days after delivery, the patient successfully underwent a mechanical aortic valve replacement and root reconstruction. In this case report, medical, surgical, and anesthetic management of parturient patients with BAV are discussed. The management of this congenital valvulopathy and vasculopathy is complicated by the extensive hemodynamic and cardiovascular derangements that occur during pregnancy.

Cone-Beam CT image contrast and attenuation-map linearity improvement (CALI) for brain stereotactic radiosurgery procedures.

A Contrast and Attenuation-map Linearity Improvement (CALI) framework is proposed for cone-beam CT (CBCT) images used for brain stereotactic radiosurgery (SRS). The proposed framework is tailored to improve soft tissue contrast of a new point-of-care image-guided SRS system that employs a challenging half cone beam geometry, but can be readily reproduced on any CBCT platform. CALI includes a pre- and post-processing step. In pre-processing we apply a shading and beam hardening artifact correction to the projections, and in post-processing step we correct the dome/capping artifact on reconstructed images caused by the spatial variations in X-ray energy generated by the bowtie-filter. The shading reduction together with the beam hardening and dome artifact correction algorithms aim to improve the linearity and accuracy of the CT-numbers (CT#). The CALI framework was evaluated using CatPhan to quantify linearity, contrast-to-noise (CNR), and CT# accuracy, as well as subjectively on patient images acquired on a clinical system. Linearity of the reconstructed attenuation-map was improved from 0.80 to 0.95. The CT# mean absolute measurement error was reduced from 76.1 to 26.9 HU. The CNR of the acrylic insert in the sensitometry module was improved from 1.8 to 7.8. The resulting clinical brain images showed substantial improvements in soft tissue contrast visibility, revealing structures such as ventricles which were otherwise undetectable in the original clinical images obtained from the system. The proposed reconstruction framework also improved CT# accuracy compared to the original images acquired on the system. For frameless image-guided SRS, improving soft tissue visibility can facilitate evaluation of MR to CBCT co-registration. Moreover, more accurate CT# may enable the use of CBCT for daily dose delivery measurements.

Synthesis of azasilacyclopentenes and silanols via Huisgen cycloaddition-initiated C-H bond insertion cascades.

An unusual transition metal-free cascade reaction of alkynyl carbonazidates was discovered to form azasilacyclopentenes. Mild thermolysis afforded the products via a series of cyclizations, rearrangements, and an α-silyl C-H bond insertion (rather than the more common Wolff rearrangement, 1,2-shift, or ß-silyl C-H insertion) to form silacyclopropanes. A mechanistic proposal for the sequence was informed by control experiments and the characterization of reaction intermediates. The substrate scope and post-cascade transformations were also explored.

Simultaneous deblurring and iterative reconstruction of CBCT for image guided brain radiosurgery.

One of the limiting factors in cone-beam CT (CBCT) image quality is system blur, caused by detector response, x-ray source focal spot size, azimuthal blurring, and reconstruction algorithm. In this work, we develop a novel iterative reconstruction algorithm that improves spatial resolution by explicitly accounting for image unsharpness caused by different factors in the reconstruction formulation. While the model-based iterative reconstruction techniques use prior information about the detector response and x-ray source, our proposed technique uses a simple measurable blurring model. In our reconstruction algorithm, denoted as simultaneous deblurring and iterative reconstruction (SDIR), the blur kernel can be estimated using the modulation transfer function (MTF) slice of the CatPhan phantom or any other MTF phantom, such as wire phantoms. The proposed image reconstruction formulation includes two regularization terms: (1) total variation (TV) and (2) nonlocal regularization, solved with a split Bregman augmented Lagrangian iterative method. The SDIR formulation preserves edges, eases the parameter adjustments to achieve both high spatial resolution and low noise variances, and reduces the staircase effect caused by regular TV-penalized iterative algorithms. The proposed algorithm is optimized for a point-of-care head CBCT unit for image-guided radiosurgery and is tested with CatPhan phantom, an anthropomorphic head phantom, and 6 clinical brain stereotactic radiosurgery cases. Our experiments indicate that SDIR outperforms the conventional filtered back projection and TV penalized simultaneous algebraic reconstruction technique methods (represented by adaptive steepest-descent POCS algorithm, ASD-POCS) in terms of MTF and line pair resolution, and retains the favorable properties of the standard TV-based iterative reconstruction algorithms in improving the contrast and reducing the reconstruction artifacts. It improves the visibility of the high contrast details in bony areas and the brain soft-tissue. For example, the results show the ventricles and some brain folds become visible in SDIR reconstructed images and the contrast of the visible lesions is effectively improved. The line-pair resolution was improved from 12 line-pair/cm in FBP to 14 line-pair/cm in SDIR. Adjusting the parameters of the ASD-POCS to achieve 14 line-pair/cm caused the noise variance to be higher than the SDIR. Using these parameters for ASD-POCS, the MTF of FBP and ASD-POCS were very close and equal to 0.7 mm-1 which was increased to 1.2 mm-1 by SDIR, at half maximum.

The origin and spread of a cooperative replicase in a prebiotic chemical system.

The origin of life requires the emergence of a system of autocatalytic polymers such as RNA. We consider a trans-acting replicase that catalyses replication of a template (either a copy of itself or another sequence). Our model includes alternating plus/minus strand replication where only the plus strand is a catalyst. Prebiotic chemistry generates random sequences and allows for non-catalysed, template-directed synthesis of new strands. These chemical reactions are insufficient to sustain replication, but they provide a background in which the first replicase can arise. In the well-mixed case, the minimum value of the catalytic rate parameter k for which a stable replicating state survives scales as 1/f, where f is the fraction of random sequences that are catalysts. When catalysts are rare (f→0), the replicating state is not stable in for any finite k because the replicases are overrun by parasitic templates already present in the prebiotic system, and by additional parasites created by mutation of the catalyst. In contrast, in 2d spatial simulations, the replicating state is stable for moderate k with appropriate values of the local diffusion constant. We calculate the probability of spread of the replicating state from a single isolated catalyst. This occurs in a parameter range that is narrower than that in which existing replicators are stable. The 2d model uses 'Two׳s Company' rules, where two molecules on a site may replicate, but crowding occurs when three molecules are on one site. A mean-field theory is presented which predicts the most important results of the spatial model. Our results emphasize that the origin of replication is a spatially-localized stochastic transition between a 'dead' state controlled by prebiotic chemistry and a 'living' state controlled by autocatalytic replication.

Noise performance of phase-insensitive multicasting in multi-stage parametric mixers.

Noise properties of large-count spectral multicasting in a phase-insensitive parametric mixer were investigated. Scalable multicasting was achieved using two-tone continuous-wave seeded mixers capable of generating more than 20 frequency non-degenerate copies. The mixer was constructed using a multistage architecture to simultaneously manage high Figure-of-Merit frequency generation and suppress noise generation. The performance was characterized by measuring the conversion efficiency and noise figure of all signal copies. Minimum noise figure of 8.09dB was measured. Experimental findings confirm that noise of the multicasted signal does not grow linearly with copy count and that it can be suppressed below this limit.