Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity

HDAC6 Regulates Mitochondrial Transport in Hippocampal Neurons

Background: Tubulin is a major substrate of the cytoplasmic class II histone deacetylase HDAC6. Inhibition of HDAC6 results in higher levels of acetylated tubulin and enhanced binding of the motor protein kinesin-1 to tubulin, which promotes transport of cargoes along microtubules. Microtubule-dependent intracellular trafficking may therefore be regulated by modulating the activity of HDAC6. We have shown previously that the neuromodulator serotonin increases mitochondrial movement in hippocampal neurons via the Akt-GSK3b signaling pathway. Here, we demonstrate a role for HDAC6 in this signaling pathway. Methodology/Principal Findings: We found that the presence of tubacin, a specific HDAC6 inhibitor, dramatically enhanced mitochondrial movement in hippocampal neurons, whereas niltubacin, an inactive tubacin analog, had no effect. Compared to control cultures, higher levels of acetylated tubulin were found in neurons treated with tubacin, and more kinesin-1 was associated with mitochondria isolated from these neurons. Inhibition of GSK3b decreased cytoplasmic deacetylase activity and increased tubulin acetylation, whereas blockade of Akt, which phosphorylates and down-regulates GSK3b, increased cytoplasmic deacetylase activity and decreased tubulin acetylation. Concordantly, the administration of 5-HT, 8-OH-DPAT (a specific 5-HT1A receptor agonist), or fluoxetine (a 5-HT reuptake inhibitor) increased tubulin acetylation. GSK3b was found to co-localize with HDAC6 in hippocampal neurons, and inhibition of GSK3b resulted in decrease

Optimization via Rejection-Free Partial Neighbor Search

Simulated Annealing using Metropolis steps at decreasing temperatures is widely used to solve complex combinatorial optimization problems. In order to improve its efficiency, we can use the Rejection-Free version of the Metropolis algorithm, which avoids the inefficiency of rejections by considering all the neighbors at every step. As a solution to avoid the algorithm from becoming stuck in local extreme areas, we propose an enhanced version of Rejection-Free called Partial Neighbor Search (PNS), which only considers random parts of the neighbors while applying Rejection-Free. We demonstrate the superior performance of the Rejection-Free PNS algorithm by applying these methods to several examples, such as the QUBO question, the Knapsack problem, the 3R3XOR problem, and the quadratic programming.Comment: 24 pages with 2 more pages of reference, 9 figure

CRISPR-Cas9 mediated cell line engineering of apoptosis pathways increases antibody expression with site-specific modifications for antibody drug conjugation

New generation of antibody drug conjugates (ADCs) have expanded the repertoire of antibody drugs in the clinic and the market for cancer and inflammation indications by using highly stable linkers to attach potent small-molecule drug to various targeting antibodies. The drug and site of drug linkage to the antibody can have profound impact on the physiochemical properties and pharmacological profile of the ADC. Ambrx has developed a technology, Eukaryotic Chemical Orthogonal Directed Engineering (EuCODE), which allows non-natural amino acids with diverse physicochemical and biological properties to be genetically encoded and site-specifically incorporated into proteins/antibodies in mammalian cells. The non-natural amino acid provides a handle for the attachment of a small-molecule drug to generate homogenous ADC with a defined Drug-to-Antibody Ratio (DAR). To establish a CHO expression system for high production of monoclonal antibodies (mAbs) containing non-natural amino acids, we successfully generated a EuCODE platform cell line stably expressing engineered amber suppressor tRNA and its cognate tRNA synthetase specific for non-natural amino acid para-acetyl phenylalanine (pAF). When transfected with antibody of interest engineered with amber nonsense codon (TAG) at selected sites suitable for drug conjugation, this EuCODE platform cell line generates stable cell lines producing pAF containing mAbs for site-specifically conjugated ADC. In order to improve production titers of pAF containing antibody and achieve a robust platform, the platform cell line and stable cell lines were further evolved using CRISPR/Cas9 genome editing technology to sequentially knock out selected genes in glutamine synthesis and apoptosis pathways to improve selection efficiency and prevent loss of viable cell mass in production cultures, respectively. Inhibition of apoptosis pathway leads to dramatic increase in viable cell mass and results in extended production time and increased productivity. Phenotypic and genetic properties of these CRISPR engineered cell lines and product quality of the antibody will be discussed in the context of using the platform to develop a commercial manufacturing cell line

Dopamine Inhibits Mitochondrial Motility in Hippocampal Neurons

The trafficking of mitochondria within neurons is a highly regulated process. In an earlier study, we found that serotonin (5-HT), acting through the 5-HT1A receptor subtype, promotes axonal transport of mitochondria in cultured hippocampal neurons by increasing Akt activity, and consequently decreasing glycogen synthase kinase (GSK3beta) activity. This finding suggests a critical role for neuromodulators in the regulation of mitochondrial trafficking in neurons. In the present study, we investigate the effects of a second important neuromodulator, dopamine, on mitochondrial transport in hippocampal neurons.Here, we show that dopamine, like 5-HT, regulates mitochondrial motility in cultured hippocampal neurons through the Akt-GSK3beta signaling cascade. But, in contrast to the stimulatory effect of 5-HT, administration of exogenous dopamine or bromocriptine, a dopamine 2 receptor (D2R) agonist, caused an inhibition of mitochondrial movement. Moreover, pretreatment with bromocriptine blocked the stimulatory effect of 5-HT on mitochondrial movement. Conversely, in cells pretreated with 5-HT, no further increases in movement were observed after administration of haloperidol, a D2R antagonist. In contrast to the effect of the D2R agonist, addition of SKF38393, a dopamine 1 receptor (D1R) agonist, promoted mitochondrial transport, indicating that the inhibitory effect of dopamine was actually the net summation of opposing influences of the two receptor subtypes. The most pronounced effect of dopamine signals was on mitochondria that were already moving directionally. Western blot analysis revealed that treatment with either a D2R agonist or a D1R antagonist decreased Akt activity, and conversely, treatment with either a D2R antagonist or a D1R agonist increased Akt activity.Our observations strongly suggest a role for both dopamine and 5-HT in regulating mitochondrial movement, and indicate that the integrated effects of these two neuromodulators may be important in determining the distribution of energy sources in neurons

The cAMP pathway is a necessary inductive signal in sympathoadrenal (SA) cell development

In primary neural crest cultures, the cAMP signaling pathway modulates both positive and negative signals influencing the development of SympathoAdrenal lineage (SA) cells. However, the mechanism by which cAMP acts as a rheostat of SA cell differentiation remains to be determined. Specifically, moderate activation of CAMP signaling promotes, in synergy with BMP2, SA cell development and the transcription of the SA lineage-determining gene Phox2a. Herein, evidence is provided that all the cAMP signaling components participate in the synergistic interaction with the BMP2 pathway in effecting SA cell development. Utilizing avian retroviral constructs, including A-CREB (dominant negative CREB), E1A and ΔE1A (a CBP interfering protein and its inactive control respectively), the role of both CBP and CREB is demonstrated in SA cell development. In addition, use of the constitutively active CREBDIEDML enabled the identification of the requirement of PKA activation in the process of SA cell development, by monitoring the expression of the proneural gene phox2a and the SA cell differentiation marker, tyrosine hydroxylase (TH). Specifically, PKA activation mediates the activation of a ser/thr phosphatase, protein phosphatase 2A (PP2A), required for activation of the proneural phox2 transcription factors. Analyses of the effect of A-CREB, CREBDIEDML, E1A and ΔE1A by clonal assays demonstrate that they do not alter neural crest cell survival, supporting that these cAMP signaling components, CREB, CBP and PKA, play an inductive role in promoting the development of neural crest cells into the SA lineage cells. This study identified, for the first time, the inductive role of the cAMP pathway in SA cell development